Expression systems

ABSTRACT

The invention relates to an expression system comprising polynucleotides encoding proteins, wherein the expression system comprises a first polynucleotide encoding at least one protein, peptide or variant thereof, which induces a T cell response, and a second polynucleotide encoding at least one protein, peptide or variant thereof, which induces an anti-pathogenic B cell response. The invention further relates to protein mixtures encoded by the expression system and cells comprising the expression system or the protein mixture and pharmaceutical compositions comprising the expression system or the protein mixture.

FIELD OF THE INVENTION

The invention relates to an expression system comprising polynucleotidesencoding proteins, wherein the expression system comprises a firstpolynucleotide encoding at least one protein, peptide or variantthereof, which induces a T cell response, and a second polynucleotideencoding at least one protein peptide or variant thereof, which inducesan anti-pathogenic B cell response. The invention further relates toprotein mixtures encoded by the expression system and cells comprisingthe expression system or the protein mixture and pharmaceuticalcompositions comprising the expression system or the protein mixture.The expression system, polynucleotides, proteins, cells, andpharmaceutical compositions are useful in the prophylaxis or treatmentof infections. The invention further relates to nucleotide constructsand expression systems encoding a modified influenza hemagglutinin (HA).

BACKGROUND OF THE INVENTION

Infectious diseases are still a major thread of mankind. One way forpreventing or treating infectious diseases is the artificial inductionof an immune response by vaccination which is the administration ofantigenic material to an individual such that an adaptive immuneresponse against the respective antigen is developed. The antigenicmaterial may be pathogens (e.g. microorganisms or viruses) which arestructurally intact but inactivated (i.e. non-infective) or which areattenuated (i.e. with reduced infectivity), or purified components ofthe pathogen that have been found to be highly immunogenic. Anotherapproach for inducing an immune response against a pathogen is theprovision of expression systems comprising one or more vector encodingimmunogenic proteins or peptides of the pathogen. Such vector may be inthe form of naked plasmid DNA, or the immunogenic proteins or peptidesare delivered by using viral vectors, for example on the basis ofmodified vaccinia viruses (e.g. Modified Vaccinia Ankara; MVA) oradenoviral vectors. Such expression systems have the advantage ofcomprising well-characterized components having a low sensitivityagainst environmental conditions.

It is a particular aim when developing vector based expression systemsthat the application of these expression systems to a patient elicits animmune response which is protective against the infection by therespective pathogen. However, although inducing an immunogenic responseagainst the pathogen, some expression systems are not able to elicit animmune response which is strong enough to fully protect againstinfections by the pathogen. Accordingly, there is still a need forexpressions systems which are capable of inducing a protective immuneresponse against a pathogen, e.g. an infectious agent like a virus.

Viruses

Viruses are a group of pathogens/infectious agents which have no ownmetabolism and can be considered as obligatory endoparasites of therespective host cells using at least parts of host's cell facilities forconducting viral protein expression and virus replication. Viruses canbe classified on the basis of the type (DNA/RNA), the strandedness(single-stranded (ss) or double-stranded (ds)), the sense (negativesense or positive sense) of the nucleic acid constituting their genomeand their replication (Baltimore classification). Accordingly, virusesare generally classified in DNA and RNA viruses. Viruses can be furtherclassified into single-stranded (ss) or double-stranded (ds) DNA or RNAviruses, which genome is a single-stranded or double-stranded nucleicacid. Some viruses have a genome which is partially double-stranded andpartially single-stranded (e.g. hepadnaviruses). The orientation or“sense” of the genome and/or in the manufacturing of medicament for usein the prophylaxis or treatment of a pathogen and/or for use in methodsof prophylaxis or treatment of a pathogen, wherein the pathogen plays animportant role for the viral life cycle of viruses, in particular in thelife cycle of ssRNA viruses or ssDNA viruses. A positive sense ssRNA (+)genome has the same orientation as a cellular mRNA and can be directlytranslated into viral proteins. In the life cycle of viruses having anegative sense single-stranded RNA genome (ssRNA (−)), it is necessarythat the genomic sequences are transcribed into positive sense mRNAwhich can be translated into viral proteins by the host cell. Asingle-stranded genome that contains both positive-sense andnegative-sense is called “ambisense” (e.g. ssRNA (+/−), ssDNA(+/−)).

Although the genome of viruses may be quite large (e.g. in the case ofDNA viruses), in particular small RNA viruses have evolutionarydeveloped strategies for expressing their gene products (e.g. proteinsand peptides) in a very efficient manner. One of these strategies is theexpression of one or more polyprotein encoded by the viral genome, whichis co- or posttranslationally processed into single proteins and/orpeptides. This strategy is adapted, for example, by some double-stranded(ds) RNA viruses or single-stranded (ss) RNA viruses having a positivesense genome. “Enveloped viruses”, such as orthomyxoviruses,paramyxoviruses, retroviruses, flaviviruses, rhabdoviruses andalphaviruses, are surrounded by a lipid bilayer originating from thehost plasma membrane (1).

Attachment glycoproteins are found in all enveloped viruses and mediatethe initial interaction between the viral envelope and the plasmamembrane of the host cell via their binding to carbohydrate moieties orcell adhesion domains of proteins or other molecules on the virus andthe membrane of the host cell. Attachment glycoproteins designated as“H” possess hemagglutinin activity, glycoproteins designated as “HN”possesses hemagglutinin and neuraminidase activities. Attachmentglycoproteins are designated as “G” when they have neitherhaemagglutination nor neuraminidase activity.

Paramyxoviruses

Paramyxoviruses are a family of animal viruses which comprises a singlestranded non-segmented negative-sense RNA. Paramyxoviruses areresponsible for a number of animal and human diseases. The RNA genome ofparamyxoviruses is 15-19 kilo bases (kb) in length and encodes 6-10genes. Each gene contains transcription start/stop signals at thebeginning and end, which are transcribed as part of the gene. The genesequence is conserved across the paramyxoviruses due to a phenomenonknown as transcriptional polarity in which genes closest to the 3′ endof the genome are transcribed in greater abundance than those towardsthe 5′ end.

After each gene is transcribed, the RNA-Dependent RNA polymerase pausesto release the new mRNA when it encounters an intergenic sequence. Whenthe RNA polymerase is paused, there is a chance that it will dissociatefrom the RNA genome. If it dissociates, it must reenter the genome atthe leader sequence, rather than continuing to transcribe the length ofthe genome. As a result, the further downstream genes are from theleader sequence, the less they will be transcribed by the RNApolymerase. The genes of paramyxoviruses are arranged in relative orderof protein needed for successful infection. The conserved gene sequenceis Nucleocapsid-Phosphoprotein-Matrix-Fusion-Attachment-Large(polymerase).

Many Paramyxovirus genomes follow the so-called “Rule of Six”. Accordingto this rule, the total length of the genome is almost always a multipleof six. However, the members of the sub-family Pneumovirinae comprisingthe Respiratory Syncytial Virus (RSV) do not follow this rule.

Respiratory Syncytial Virus (RSV)

The enveloped virus designated as respiratory syncytial virus (RSV) isthe most important cause of viral lower respiratory tract illness (LRTI)in infants and children worldwide (2). In the United States, it isestimated that 70,000-126,000 infants are hospitalized annually with RSVpneumonia or bronchiolitis and that the rate of hospitalization forbronchiolitis has increased since 1980 (3). Children are infected by 2years of age and the WHO has estimated that RSV causes disease inapproximately 64 million children each year and 160,000 deaths. Inindustrialised countries, RSV is responsible for at least 50% ofhospitalisations for respiratory disease in children, and up to 6% ofall RSV infections in children result in hospitalisation (4). RSVinfection does not provoke lasting immunity, so that human hostsexperience lifelong cycles of infection and re-infection. Although it istraditionally regarded as a pediatric pathogen, RSV also causes severedisease in the elderly and immuno-compromised individuals (5). Theburden of RSV disease in the elderly is comparable to that of seasonalinfluenza and the economic impact of RSV-related disease in adults isestimated to be greater than that of influenza in relation to numbers ofdays lost from work (6, 7). Monoclonal antibody prophylaxis is effectivein reducing RSV hospitalisations by 50% in infants at high risk ofsevere disease (8). However, there is currently no effective RSV vaccineor anti-viral therapy.

The disastrous effect of a formalin-inactivated (FI) RSV vaccine ininfants in the 1960s has hampered vaccine development. The vaccinefailed to protect against RSV infection and induced exacerbatedrespiratory disease (9) which has been attributed to induction of hightitre, poorly neutralising, low affinity antibodies, lack of CD8+ T cellpriming and induction of a Th2-biased immune response (10, 11, and 12).There is evidence that RSV impairs the induction of an adequate adaptiveT cell immune response (13).

There is, therefore, a clear need for an effective vaccine not only toprotect infants, but also to boost immunity in the elderly and to reducethe circulation of RSV in siblings and adults, who are the main sourceof RSV infection for infants. A RSV vaccine capable of inducingneutralizing antibody response and potent and broad T cell response forpriming a T cell responses in individuals who have not yet been infectedwith RSV (infants) or for boosting a pre-existing T cell response inindividuals who need to ‘reset’ the memory response to higher levels(elderly) is especially desirable.

Orthomyxoviruses

Orthomyxoviruses are a family of RNA viruses that includes five genera:Influenzavirus A, Influenzavirus B, Influenzavirus C, Isavirus andThogotovirus. A sixth genus has recently been described. The first threegenera contain viruses that cause influenza in vertebrates, includingbirds, humans, and other mammals. The three genera of influenza virushave antigenic differences in their nucleoprotein and matrix protein.Influenzavirus A infects humans, other mammals, and birds, and causesall influenza pandemics. Influenzavirus B infects humans and seals.Influenzavirus C infects humans and pigs.

Viruses of the Orthomyxovirus family contain 6 to 8 segments of linearnegative-sense single stranded RNA. The total genome length is12000-15000 nucleotides (nt). The largest segment 2300-2500 nt; ofsecond largest 2300-2500 nt; of third 2200-2300 nt; of fourth 1700-1800nt; of fifth 1500-1600 nt; of sixth 1400-1500 nt; of seventh 1000-1100nt; of eighth 800-900 nt. Genome sequence has terminal repeatedsequences; repeated at both ends. Terminal repeats at the 5′-end are12-13 nucleotides long. The nucleotide sequences of 3′-terminus areidentical the same in genera of same family; most on RNA (segments), oron all RNA species. Terminal repeats at the 3′-end 9-11 nucleotideslong.

Influenza virus is one of the most important respiratory pathogens. Inthe US alone, influenza infection is responsible for 20,000-40,000deaths and over 100,000 hospitalizations annually (1). Infants, theelderly, and individuals with compromised cardiac, pulmonary, or immunesystems are at great risk of serious complications following fluinfection.

Immunization proves to be the most effective measure in preventing thedisease. One of the common features shared by all current influenzavaccines consists in targeting primarily the induction of neutralizingantibodies directed against the major viral envelope protein,hemagglutinin (HA).

SUMMARY OF THE INVENTION

The invention provides in a first aspect an expression system comprisinga first polynucleotide encoding at least one protein, peptide or variantthereof, which induces a T cell response and a second polynucleotideencoding at least one protein, peptide or variant thereof, which inducesan anti-pathogenic B cell response.

In a second aspect, the invention provides an isolated protein mixtureencoded by the expression system of the first aspect.

In a third aspect, the invention provides an isolated host cellcontaining the expression system of the first aspect and/or the proteinmixture of the second aspect.

In a fourth aspect, the present invention provides a compositioncomprising the expression system of the first aspect, or the proteinmixture of the second aspect, and a pharmaceutical acceptable carrierand/or excipient.

In a fifth aspect, the present invention provides the expression systemof the first aspect, the protein mixture of the second aspect, the cellof the third aspect and the composition of the fourth aspect, for theuse in medicine in particular in the treatment or prevention ofinfectious diseases, preferably a viral disease.

In a sixth aspect, the present invention provides for a method oftreatment or prevention of a viral disease comprising the administrationof an effective amount of the expression system of the first aspect, theprotein mixture of the second aspect, the cell of the third aspect andthe composition of the fourth aspect.

In a seventh aspect, the present invention provides for a method ofenhancing an immune response comprising the administration of theexpression system of the first aspect, the protein mixture of the secondaspect, the cell of the third aspect and the composition of the fourthaspect.

In an eighth aspect, the present invention provides nucleotideconstructs encoding influenza hemagglutinin (HA), an expression systemcomprising these nucleotide constructs, and proteins or polyproteinsencoded by the nucleotide constructs or the expression system, whereinthe HA0 cleavage site has a multibasic sequence.

In a ninth aspect, the present invention provides the use of themultibasic HA0 cleavage site for constructing expression systems capablefor expressing influenza hemagglutinin (HA) in vitro and/or in vivo.

In a tenth aspect, the invention provides an isolated protein mixtureencoded by the expression system of the eighth aspect.

In an eleventh aspect, the invention provides an isolated host cellcontaining the nucleotide constructs, the expression system or theproteins or polyproteins of the eighth aspect and/or the protein mixtureof the tenth aspect.

In a twelfth aspect, the present invention provides a compositioncomprising the nucleotide constructs, the expression system or theproteins or polyproteins of the eighth aspect, or the protein mixture ofthe tenth aspect, and a pharmaceutical acceptable carrier and/orexcipient.

In a thirteenth aspect, the present invention provides the nucleotideconstructs, the expression system or the proteins or polyproteins of theeighth aspect, the protein mixture of the tenth aspect, the cell of theeleventh aspect and the composition of the twelfth aspect, for the usein medicine in particular in the treatment or prevention of influenzavirus infections.

In a fourteenth aspect, the present invention provides for a method oftreatment or prevention of an influenza virus infections comprising theadministration of an effective amount of the nucleotide constructs, theexpression system or the proteins or polyproteins of the eighth aspect,the protein mixture of the tenth aspect, the cell of the eleventh aspectand the composition of the twelfth aspect.

In a fifteenth aspect, the present invention provides for a method ofenhancing an immune response comprising the administration of thenucleotide constructs, the expression system or the proteins orpolyproteins of the eighth aspect, the protein mixture of the tenthaspect, the cell of the eleventh aspect and the composition of thetwelfth aspect.

The above summary does not necessarily describe all aspects of thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic Diagram of the RSV vaccine polyprotein.conF0=consensus sequence of the F protein, 2A=translational cleavagesite of the Foot and Mouth Disease virus, conN=consensus sequence of theN protein, conM2-1=consensus of the M2-1 protein.

FIG. 2: The vaccine antigen F0ΔTM-N-M2-1 is efficiently processed inmammalian cells. Western Blot analysis of lysates from HeLa cells; nt:not transfected Hela. RSV transf: HeLa cells transfected withF0ΔTM-N-M2-1. RSV inf: Hep2 cells infected with RSV strain A

FIG. 3: The secreted F protein forms a homotrimer. Western Blot analysisof supernatant from transfected HeLa cells; RSV: F0ΔTM-N-M2-1transfected, F0: F0ΔTM transfected, Ctrl: empty plasmid transfected

FIG. 4: The F protein expressed from the vaccine polyprotein is a betterimmunogen than the F protein alone. A. Western Blot analysis ofsupernatant from HeLa cells infected with PanAd3/F0ΔTM-N-M2-1 which wasprobed with different dilutions of sera from mice immunized with F0ΔTMor F0ΔTM-N-M2-1 B. densitometric scanning of the Western Blot on panelA. Data are expressed as Relative Intensity of the area corresponding tothe protein band.

FIG. 5: The RSV vaccine induced potent systemic T cell immunity in miceby a single intramuscular injection. IFNg-Elispot assay of splenocytesof PanAd3/F0ΔTM-N-M2-1 immunized Balb/C mice using mapped immunodominantpeptides from RSV F and M proteins.

FIG. 6: Schematic diagram of the Influenza vaccine polyprotein.NP=consensus sequence of the NP protein, M1=consensus sequence of M1protein, 2A=translational cleavage site of the Foot and Mouth Diseasevirus, H1p=consensus sequence of the HA protein from H1N12009.

FIG. 7 Western Blot analysis of H1p expression in transfected HeLacells. Total lysate of HeLa cells transfected with PVJ-H1p (Lane 1),with PVJ-H1 (Lane 2) and not transfected CTR (Lane 3). The arrows showthe bands corresponding to the uncleaved (70kD) HA0 form and the cleaved(28 Kd) HA2. The polyclonal anti-HA serum recognize epitopes in the HA2protein fragment. It is shown that the H1p protein is fully processed.

FIG. 8 Whole-cell FACS analysis of membrane-displayed HA proteins. Thehistograms represent the median fluorescence analysis of HeLa cellstransfected with wild type HA (right upper panel) and H1 (right lowerpanel). Cells were incubated with hyperimmune mouse polyclonal serumraised against H1p and then with a secondary anti-mouse antibodyPE-conjugated. In the left upper and lower panels, cells were incubatedwith mouse pre-immune serum to set the background fluorescence level.

FIG. 9 H1p is able to induce higher antibody titers. ELISA assay oncoated recombinant HA (H1N1California 2009). Antibody titers weremeasured on sera from animals immunized with H1 and H1p. Titers werecalculated by serial dilution of the sera and represents the dilutiongiving an OD value three times higher than the background.

FIG. 10 HA (H1N1Mexico2009) pseudotyped virus infection of MDCK cells ismore potently neutralized by the serum of animals immunized with H1p.Results of an ELISA assay on coated recombinant HA (H1N1California 2009)with the sera of animals immunized with H1p and NPM1H1p.

FIG. 11 H1p expressed in the context of the triple antigen is able toinduce higher antibody titers. ELISA assay on coated recombinant HA(H1N1California 2009). Antibody titers were measured on sera fromanimals immunized with H1p and NPM1H1p. Titers were calculated by serialdilution of the sera and represent the dilution giving an OD value threetimes higher than the background.

FIG. 12 Western Blot analysis of NPM1H1p antigen expression intransfected HeLa cells shows that the protein is fully processed. Totallysate of HeLa cells transfected with pNEB-NPM1H1p (Lane 1), withpNEB-NPM1 (Lane 2) and mock transfected (Lane 3). The arrow shows theband corresponding to the fusion protein NPM1 (70kD. A monoclonalanti-NP antibody has been used to detect the intracellular protein.

FIG. 13 H1p derived from processing of NPM1H1p is displayed on the cellmembrane and correctly folded. Whole-cell FACS analysis of HeLa cellsmock transfected (left panel) or transfected with NPM1H1p (right panel).Cells were incubated with the mouse mAb C179 which binds to aconformational epitope in the HA stem region and then with a secondaryanti-mouse antibody PE-conjugated.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, GenBank Accession Number sequence submissions etc.),whether supra or infra, is hereby incorporated by reference in itsentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

DEFINITIONS

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The abbreviations “F” or “F0” are used interchangeably herein and referto the Fusion protein of paramyxoviruses, preferably of RSV.

The abbreviation “G” refers to the Glycoprotein of paramyxoviruses,preferably of pneumovirinae, more preferably of RSV.

The abbreviation “H” refers to the Hemagglutinin Protein ofparamyxoviruses, preferably of morbilliviruses.

The abbreviation “HN” refers to the Hemagglutinin-Neuraminidase Proteinof paramyxoviruses, particularly of Respirovirus, Avulavirus andRubulavirus.

The abbreviation “N” refers to the Nucleocapsid protein ofparamyxoviruses, preferably of RSV.

The abbreviation “M” refers to the glycosylated Matrix protein ofparamyxoviruses, preferably of RSV.

With respect to paramyxoviruses, the abbreviation “M2” or “M2-1” refersto the non-glycosylated Matrix protein of paramyxoviruses, preferably ofRSV.

The abbreviation “P” refers to the Phosphoprotein of paramyxoviruses,preferably of RSV.

With respect to paramyxoviruses, the abbreviation “NS1” and “NS2” referto the non-structural proteins 1 and 2 of paramyxoviruses, preferably ofRSV.

The abbreviation “L” refers to the catalytic subunit of the polymeraseof paramyxoviruses, preferably of RSV.

The abbreviation “HA” refers to the hemagglutinin of orthomyxovirus,preferably influenzaviruses, more preferably of influenza A virus.

The abbreviation “HA0” refers to the precursor protein of hemagglutininsubunits HA1 and HA2 of orthomyxovirus, preferably influenzaviruses,more preferably of influenza A virus

The abbreviation “H1p” refers to the modified hemagglutinin oforthomyxovirus, preferably influenzaviruses, more preferably ofinfluenza A virus.

The abbreviation “NA” refers to the neuraminidase of orthomyxovirus,preferably influenzaviruses, more preferably of influenza A virus.

The abbreviation “NP” refers to the nucleoprotein of orthomyxoviruses,preferably influenzaviruses, more preferably of influenza A virus.

The abbreviation “M1” refers to the matrixprotein 1 of orthomyxoviruses,preferably influenzaviruses, more preferably of influenza A virus.

With respect to orthomyxoviruses, the abbreviation “M2” refers to theMatrix protein M2 of orthomyxoviruses, preferably influenzaviruses, morepreferably of influenza A virus.

With respect to orthomyxovirus, the abbreviation “NS1” refers to thenon-structural protein 1 of orthomyxoviruses, preferablyinfluenzaviruses, more preferably of influenza A virus.

The abbreviation “NS2/NEP” refers to the non-structural protein 2 (alsoreferred to as NEP, nuclear export protein) of orthomyxoviruses,preferably influenzaviruses, more preferably influenza A virus.

The abbreviation “PA” refers to a polymerase subunit protein oforthomyxoviruses, preferably influenzaviruses, more preferably influenzaA virus.

The abbreviation “PB1” refers to a polymerase subunit protein oforthomyxoviruses, preferably influenzaviruses, more preferably influenzaA virus.

The abbreviation “PB2” refers to a polymerase subunit protein oforthomyxoviruses, preferably influenzaviruses, more preferably influenzaA virus.

The abbreviation “PB1-F2” or “PB1F2” refers to a protein encoded by analternate reading frame in the PB1 Gene segment of orthomyxoviruses,preferably influenzaviruses, more preferably influenza A virus.

The term “expression system” as used herein refers to a system designedto produce one or more gene products of interest. Typically, such systemis designed “artificially”, i.e. by gene-technological means usable toproduce the gene product of interest either in vitro in cell-freesystems or in vivo in cell-based systems. It is understood thatnaturally occurring expression systems such as for instance nativeviruses are not encompassed by the expression system of the presentinvention.

The “gene product of interest” typically refers to a macromolecule suchas but not limited to RNA, peptide, polypeptide, or protein, or segment,epitope, or fragment thereof.

In an expression system the gene product of interest is encoded for byone or more nucleic acid molecules. Nucleic acid molecules areunderstood as a polymeric macromolecules made from nucleotide monomers.Nucleotide monomers are composed of a nucleobase, a five-carbon sugar(such as but not limited to ribose or 2′-deoxyribose), and one to threephosphate groups. Typically, a polynucleotide is formed throughphosphodiester bonds between the individual nucleotide monomers. In thecontext of the present invention referred to nucleic acid moleculesinclude but are not limited to ribonucleic acid (RNA) anddeoxyribonucleic acid (DNA). The terms “polynucleotide” and “nucleicacid” are used interchangeably herein.

In cell-free expression systems isolated polynucleotides are used astemplate for in vitro translation reactions. In cell-based expressionsystems polynucleotides are comprised on one or more vectors. As usedherein, the term “vector” refers to a protein or a polynucleotide or amixture thereof which is capable of being introduced or of introducingthe proteins and/or nucleic acid comprised therein into a cell. In thecontext of the present invention it is preferred that the genes ofinterest encoded by the introduced polynucleotide are expressed withinthe cell upon introduction of the vector or vectors. Examples ofsuitable vectors include but are not limited to plasmids, cosmids,phages, viruses or artificial chromosomes.

The phrase “induction of T cell response” refers to the generation orthe re-stimulation of virus specific CD4+ or CD8+ T cells. Theexpression system of the invention can induce or re-stimulate a T cellmediated adaptive response directed to the MHC class I or class IIepitopes present in the viral proteins expressed by the polynucleotide.Such T cell response can be measured by art known methods, preferably byex-vivo re-stimulation of T cells with synthetic peptides spanning theentire viral proteins and analysis of proliferation or Interferon-gammaproduction.

The phrase “induction of B cell response” refers to the generation orthe re-stimulation of virus specific B cells producing immunoglobulinsof class IgG or IgA. The expression system of the invention can induceor re-stimulate B cells producing antibodies specific for pathogenic,e.g. viral, antigens expressed by the polynucleotide. Such B cellresponse can be measured by ELISA (Enzyme Linked Immuno Stained Assay)assay with the synthetic antigen of serum or mucosal immunoglobulin.Alternatively the induced antibody titer can be measured by virusneutralization assays.

The phrase “induction of an anti-pathogenic B cell response” refers tothe generation or the re-stimulation of virus specific B cells producingimmunoglobulins of class IgG or IgA which inactivates, eliminates,blocks and/or neutralizes the respective pathogen such that the diseasecaused by the pathogen does not break out and/or the symptoms arealleviated. This is also called a “protective immune response” againstthe pathogen. The expression system of the invention can induce orre-stimulate B cells producing antibodies specific for pathogenic, e.g.viral, antigens expressed by the polynucleotide. Such B cell responsecan be measured by ELISA (Enzyme Linked Immuno Stained Assay) assay withthe synthetic antigen of serum or mucosal immunoglobulin. Alternativelythe induced antibody titer can be measured by virus neutralizationassays.

The phrase “enhancing an immune response” refers to the strengthening orintensification of the humoral and/or cellular immune response againstan immunogen, preferably pathogens, more preferably viruses. Theenhancement of the immune response can be measured by comparing theimmune response elicited by an expression system of the invention withthe immune response of an expression system expressing the sameantigen/immunogen alone by using tests described herein and/or testswell known in the present technical field.

In an expression system, a gene of interest may be encoded by a singlepolynucleotide or by several separate polynucleotides. In cell-basedexpression systems one or more polynucleotides may be comprised on asingle or on several separate vectors. Each of these polynucleotides mayencode the whole or a part of the gene product of interest.

Furthermore, expression systems may encompass “expression controlsequences” that regulate the expression of the gene of interest.Typically, expression control sequences are polypeptides orpolynucleotides such as but not limited to promoters, enhancers,silencers, insulators, or repressors.

Accordingly, a vector comprising one or more polynucleotides encodingfor one or more gene products of interest may comprise furtherexpression control sequences. In a vector comprising more than onepolynucleotide encoding for one or more gene products of interest, theexpression may be controlled together or separately by one or moreexpression control sequences. More specifically, each polynucleotidecomprised on the vector may be control by a separate expression controlsequence or all polynucleotides comprised on the vector may becontrolled by a single expression control sequence. Polynucleotidescomprised on a single vector controlled by a single expression controlsequences preferably form an open reading frame.

The term “expression system” further encompasses the expression of thegene product of interest comprising the transcription of thepolynucleotides, RNA splicing, translation into a polypeptide, andpost-translational modification of a polypeptide or protein.

The term “open reading frame” (ORF) refers to a sequence of nucleotides,that can be translated into amino acids. Typically, such an ORF containsa start codon, a subsequent region usually having a length which is amultiple of 3 nucleotides, but does not contain a stop codon (TAG, TAA,TGA, UAG, UAA, or UGA) in the given reading frame. Typically, ORFs occurnaturally or are constructed artificially, i.e. by gene-technologicalmeans. An ORF codes for a protein where the amino acids into which itcan be translated form a peptide-linked chain.

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein and refer to any peptide-linked chain of aminoacids, regardless of length or post-translational modification.

The term “post-translational” used herein refers to events that occurafter the translation of a nucleotide triplet into an amino acid and theformation of a peptide bond to the proceeding amino acid in thesequence. Such post-translational events may occur after the entirepolypeptide was formed or already during the translation process onthose parts of the polypeptide that have already been translated.Post-translational events typically alter or modify the chemical orstructural properties of the resultant polypeptide. Examples ofpost-translational events include but are not limited to events such asglycosylation or phosphorylation of amino acids, or cleavage of thepeptide chain, e.g. by an endopeptidase.

The term “co-translational” used herein refers to events that occurduring the translation process of a nucleotide triplet into an aminoacid chain. Those events typically alter or modify the chemical orstructural properties of the resultant amino acid chain. Examples ofco-translational events include but are not limited to events that maystop the translation process entirely or interrupted the peptide bondformation resulting in two discreet translation products.

As used herein, the terms “polyprotein” or “artificial polyprotein”refer to an amino acid chain that comprises, or essentially consists ofor consists of two amino acid chains that are not naturally connected toeach other. The polyprotein may comprise one or more further amino acidchains. Each amino acid chain is preferably a complete protein, i.e.spanning an entire ORF, or a fragment, domain or epitope thereof. Theindividual parts of a polyprotein may either be permanently ortemporarily connected to each other. Parts of a polyprotein that arepermanently connected are translated from a single ORF and are not laterseparated co- or post-translationally. Parts of polyproteins that areconnected temporarily may also derive from a single ORF but are dividedco-translationally due to separation during the translation process orpost-translationally due to cleavage of the peptide chain, e.g. by anendopeptidase. Additionally or alternatively, parts of a polyprotein mayalso be derived from two different ORF and are connectedpost-translationally, for instance through covalent bonds.

Proteins or polyproteins usable in the present invention (includingprotein derivatives, protein variants, protein fragments, proteinsegments, protein epitopes and protein domains) can be further modifiedby chemical modification. This means such a chemically modifiedpolypeptide comprises other chemical groups than the 20 naturallyoccurring amino acids. Examples of such other chemical groups includewithout limitation glycosylated amino acids and phosphorylated aminoacids. Chemical modifications of a polypeptide may provide advantageousproperties as compared to the parent polypeptide, e.g. one or more ofenhanced stability, increased biological half-life, or increased watersolubility. Chemical modifications applicable to the variants usable inthe present invention include without limitation: PEGylation,glycosylation of non-glycosylated parent polypeptides, or themodification of the glycosylation pattern present in the parentpolypeptide. Such chemical modifications applicable to the variantsusable in the present invention may occur co- or post-translational.

The term “segment” refers to any part of a macromolecule (e.g. apolypeptide, protein or polyprotein) into which this macromolecule canbe divided. A macromolecule may consist of one or more segments. Suchsegmentation may exist due to functional (e.g. having immunoreactivefeatures or membrane attachment functions) or structural (e.g.nucleotide or amino acid sequence, or secondary or tertiary structure)properties of the macromolecule and/or the individual segment. In thecontext of the present invention it is preferred that the term “segment”refers to a part of a protein or polyprotein. It is particularlypreferred that such segment folds and/or functions independently of therest of the protein or polyprotein.

An “epitope”, also known as antigenic determinant, is the segment of amacromolecule that is recognized by the immune system, specifically byantibodies, B cells, or T cells. Such epitope is that part or segment ofa macromolecule capable of binding to an antibody or antigen-bindingfragment thereof. In this context, the term “binding” preferably relatesto a specific binding. In the context of the present invention it ispreferred that the term “epitope” refers to the segment of protein orpolyprotein that is recognized by the immune system. Epitopes usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and usually have specificthree-dimensional structural characteristics, as well as specific chargecharacteristics. Conformational and non-conformational epitopes aredistinguished in that the binding to the former but not the latter islost in the presence of denaturing solvents.

As used herein, the term “domain” refers to the segment of a protein orpolyprotein sequence or structure (or corresponding nucleotide sequence)that can evolve, function, and/or exist independently of the rest of theprotein chain. Typically, a protein consists of one or several domainswith each of them being three-dimensional structure that are stable andfolded independently of the rest of the protein chain. Such domaintypically forms an independent functional unit within the protein (e.g.transmembrane-domains, immunoglobulin-like domains, or DNA-bindingdomains).

As used herein, the term protein or segment “variant” is to beunderstood as a polypeptide (or segment) which differs in comparison tothe polypeptide (or segment, epitope, or domain) from which it isderived by one or more changes in the amino acid sequence. Thepolypeptide from which a protein variant is derived is also known as theparent polypeptide. Likewise, the segment from which a segment variantis derived from is known as the parent segment. Typically, a variant isconstructed artificially, preferably by gene-technological means.Typically, the parent polypeptide is a wild-type protein or wild-typeprotein domain. In the context of the present invention it is furtherpreferred that a parent polypeptide (or parent segment) is the consensussequence of two or more wild-type polypeptides (or wild-type segments).Further, the variants usable in the present invention may also bederived from homologs, orthologs, or paralogs of the parent polypeptideor from artificially constructed variant, provided that the variantexhibits at least one biological activity of the parent polypeptide. Thechanges in the amino acid sequence may be amino acid exchanges,insertions, deletions, N-terminal truncations, or C-terminaltruncations, or any combination of these changes, which may occur at oneor several sites. In preferred embodiments, a variant usable in thepresent invention exhibits a total number of up to 200 (up to 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or200) changes in the amino acid sequence (i.e. exchanges, insertions,deletions, N-terminal truncations, and/or C-terminal truncations). Theamino acid exchanges may be conservative and/or non-conservative. Inpreferred embodiments, a variant usable in the present invention differsfrom the protein or domain from which it is derived by up to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100 amino acid exchanges, preferably conservativeamino acid changes.

Alternatively or additionally, a “variant” as used herein, can becharacterized by a certain degree of sequence identity to the parentpolypeptide or parent polynucleotide from which it is derived. Moreprecisely, a protein variant in the context of the present inventionexhibits at least 80% sequence identity to its parent polypeptide. Apolynucleotide variant in the context of the present invention exhibitsat least 80% sequence identity to its parent polynucleotide. Preferably,the sequence identity of protein variants is over a continuous stretchof 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids.Preferably, the sequence identity of polynucleotide variants is over acontinuous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 ormore nucleotides.

The term “at least 80% sequence identity” is used throughout thespecification with regard to polypeptide and polynucleotide sequencecomparisons. This expression preferably refers to a sequence identity ofat least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99% tothe respective reference polypeptide or to the respective referencepolynucleotide. Preferably, the polypeptide in question and thereference polypeptide exhibit the indicated sequence identity over acontinuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or moreamino acids or over the entire length of the reference polypeptide.Preferably, the polynucleotide in question and the referencepolynucleotide exhibit the indicated sequence identity over a continuousstretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or morenucleotides or over the entire length of the reference polypeptide.

Variants may additionally or alternatively comprise deletions of aminoacids, which may be N-terminal truncations, C-terminal truncations orinternal deletions or any combination of these. Such variants comprisingN-terminal truncations, C-terminal truncations and/or internal deletionsare referred to as “deletion variant” or “fragments” in the context ofthe present application. The terms “deletion variant” and “fragment” areused interchangeably herein. A fragment may be naturally occurring (e.g.splice variants) or it may be constructed artificially, preferably bygene-technological means. Preferably, a fragment (or deletion variant)has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidsat its N-terminus and/or at its C-terminus and/or internally as comparedto the parent polypeptide, preferably at its N-terminus, at its N- andC-terminus, or at its C-terminus. In case where two sequences arecompared and the reference sequence is not specified in comparison towhich the sequence identity percentage is to be calculated, the sequenceidentity is to be calculated with reference to the longer of the twosequences to be compared, if not specifically indicated otherwise. Ifthe reference sequence is indicated, the sequence identity is determinedon the basis of the full length of the reference sequence indicated bySEQ ID, if not specifically indicated otherwise. For example, a peptidesequence consisting of 50 amino acids compared to the amino acidsequence of protein F according to SEQ ID NO: 1 may exhibit a maximumsequence identity percentage of 10.04% (50/498) while a sequence with alength of 249 amino acids may exhibit a maximum sequence identitypercentage of 50.00% (249/498).

The similarity of nucleotide and amino acid sequences, i.e. thepercentage of sequence identity, can be determined via sequencealignments. Such alignments can be carried out with several art-knownalgorithms, preferably with the mathematical algorithm of Karlin andAltschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) orwith the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T.J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. onhttp://www.ebi.ac.uk/Tools/clustalw/ or onhttp://www.ebi.ac.uk/Tools/clustalw2/index.html or onhttp://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html.Preferred parameters used are the default parameters as they are set onhttp://www.ebi.ac.uk/Tools/clustalw/ orhttp://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequenceidentity (sequence matching) may be calculated using e.g. BLAST, BLAT orBlastZ (or BlastX). A similar algorithm is incorporated into the BLASTNand BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST polynucleotide searches are performed with the BLASTNprogram, score=100, word length=12, to obtain polynucleotide sequencesthat are homologous to those nucleic acids which encode F, N, or M2-1.BLAST protein searches are performed with the BLASTP program, score=50,word length=3, to obtain amino acid sequences homologous to the Fpolypeptide, N polypeptide, or M2-1 polypeptide. To obtain gappedalignments for comparative purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs are used.

Sequence matching analysis may be supplemented by established homologymapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b,19 Suppl 1:I54-I62) or Markov random fields. When percentages ofsequence identity are referred to in the present application, thesepercentages are calculated in relation to the full length of the longersequence, if not specifically indicated otherwise.

The polynucleotides of the invention encodes proteins, peptides orvariants thereof which comprise amino acids which are designatedfollowing the standard one- or three-letter code according to WIPOstandard ST.25 unless otherwise indicated. If not indicated otherwise,the one- or three letter code is directed at the naturally occurringL-amino acids and the amino acid sequence is indicated in the directionfrom the N-terminus to the C-terminus of the respective protein, peptideor variant thereof.

“Hybridization” can also be used as a measure of sequence identity orhomology between two nucleic acid sequences. A nucleic acid sequenceencoding a protein of the invention, or a portion of any of these can beused as a hybridization probe according to standard hybridizationtechniques. The hybridization of a respective probe to DNA or RNA from atest source is an indication of the presence of the target DNA or RNA,respectively, in the test source.

Hybridization conditions are known to those skilled in the art and canbe found, for example, in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y., 6.3.1-6.3.6, 1991. “Moderate hybridizationconditions” are defined as equivalent to hybridization in 2× sodiumchloride/sodium citrate (SSC) at 30° C., followed by a wash in 1×SSC,0.1% SDS at 50° C. “Highly stringent conditions” are defined asequivalent to hybridization in 6× sodium chloride/sodium citrate (SSC)at 45° C., followed by a wash in 0.2×SSC, 0.1% SDS at 65° C.

Additionally or alternatively a deletion variant may occur not due tostructural deletions of the respective amino acids as described above,but due to these amino acids being inhibited or otherwise not able tofulfill their biological function. Typically, such functional deletionoccurs due to the insertions to or exchanges in the amino acid sequencethat changes the functional properties of the resultant protein, such asbut not limited to alterations in the chemical properties of theresultant protein (i.e. exchange of hydrophobic amino acids tohydrophilic amino acids), alterations in the post-translationalmodifications of the resultant protein (e.g. post-translational cleavageor glycosylation pattern), or alterations in the secondary or tertiaryprotein structure. Additionally or alternatively, a functional deletionmay also occur due to transcriptional or post-transcriptional genesilencing (e.g. via siRNA) or the presence or absence of inhibitorymolecules such as but not limited to protein inhibitors or inhibitoryantibodies.

In the context of the present invention it is preferred that a protein(or a segment or a domain or an epitope) being “functionally deleted”refers to the fact that the amino acids or nucleotides of thecorresponding sequence are either deleted or present but not fulfillingtheir biological function.

As used herein, the term “consensus” refers to an amino acid ornucleotide sequence that represents the results of a multiple sequencealignment, wherein related sequences were compared to each other. Suchconsensus sequence is composed of the amino acids or nucleotides mostcommonly observed at each position. In the context of the presentinvention it is preferred that the sequences used in the sequencealignment to obtain the consensus sequence are sequences of differentviral subtypes/serotypes strains isolated in various different diseaseoutbreaks worldwide. Each individual sequence used in the sequencealignment is referred to as the sequence of a particular virus“isolate”. A more detailed description of the mathematical methods toobtain such consensus is provided in the Example section. In case thatfor a given position no “consensus nucleotide” or “consensus amino acid”can be determined, e.g. because only two isolates were compared, than itis preferred that the amino acid of each one of the isolates is used.The resulting protein is assessed for its respective B cell and/or Tcell inducing ability.

A “peptide linker” (or short: “linker”) in the context of the presentinvention refers to an amino acid sequence of between 1 and 100 aminoacids. In preferred embodiments, a peptide linker according to thepresent invention has a minimum length of at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acids. In further preferred embodiments, apeptide linker according to the present invention has a maximum lengthof 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32,31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15amino acids or less. It is preferred that peptide linkers provideflexibility among the two amino acid proteins, fragments, segments,epitopes and/or domains that are linked together. Such flexibility isgenerally increased if the amino acids are small. Thus, preferably thepeptide linker of the present invention has an increased content ofsmall amino acids, in particular of glycins, alanines, serines,threonines, leucines and isoleucines. Preferably, more than 20%, 30%,40%, 50%, 60% or more of the amino acids of the peptide linker are smallamino acids. In a preferred embodiment the amino acids of the linker areselected from glycines and serines. In especially preferred embodiments,the above-indicated preferred minimum and maximum lengths of the peptidelinker according to the present invention may be combined, if such acombination makes mathematically sense. In further preferredembodiments, the peptide linker of the present invention isnon-immunogenic; in particularly preferred embodiments, the peptidelinker is non-immunogenic to humans.

The term “cleavage site” as used herein refers to an amino acid sequenceor nucleotide sequence where this sequence directs the division, e.g.because it is recognized by a cleaving enzyme, and/or can be divided.Typically, a polypeptide chain is cleaved by hydrolysis of one or morepeptide bonds that link the amino acids and a polynucleotide chain iscleaved by hydrolysis of one or more of the phosphodiester bond betweenthe nucleotides. Cleavage of peptide- or phosphodiester-bonds mayoriginate from chemical or enzymatic cleavage. Enzymatic cleavage refersto such cleavage being attained by proteolytic enzymes including but notlimited to restriction endonuclease (e.g. type I, type II, type II, typeIV or artificial restriction enzymes) and endo- or exo-peptidases or-proteases (e.g. serine-proteases, cysteine-proteases,metallo-proteases, threonine proteases, aspartate proteases, glutamicacid proteases). Typically, enzymatic cleavage occurs due toself-cleavage or is effected by an independent proteolytic enzyme.Enzymatic cleavage of a protein or polypeptide can happen either co- orpost-translational. Accordingly, the term “endopeptidase cleavage site”used herein, refers to a cleavage cite within the amino acid ornucleotide sequence where this sequence is cleaved or is cleavable by anendopeptidase (e.g. trypsin, pepsin, elastase, thrombin, collagenase,furin, thermolysin, endopeptidase V8, cathepsins). Alternatively oradditionally, the polyprotein of the present invention can be cleaved byan autoprotease, i.e. a protease which cleaves peptide bonds in the sameprotein molecule which also comprises the protease. Examples of suchautoproteases are the NS2 protease from flaviviruses or the VP4 proteaseof birnaviruses.

Alternatively, the term “cleavage site” refers to an amino acid sequenceor nucleotide sequence that prevents the formation of peptide- orphosphodiester-bonds between amino acids or nucleotides, respectively.For instance, the bond formation may be prevented due toco-translational self-processing of the polypeptide or polyproteinresulting in two discontinuous translation products being derived from asingle translation event of a single open reading frame. Typically, suchself-processing is effected by a “ribosomal skip” caused by a pseudostop-codon sequence that induces the translation complex to move fromone codon to the next without forming a peptide bond. Examples ofsequences inducing a ribosomal skip include but are not limited to viral2A peptides or 2A-like peptide (herein both are collectively referred toas “2A peptide” or interchangeably as “2A site” or “2A cleavage site”)which are used by several families of viruses, including Picornavirus,insect viruses, Aphtoviridae, Rotaviruses and Trypanosoma. Best knownare 2A sites of rhinovirus and foot-and-mouth disease virus of thePicornaviridae family which are typically used for producing multiplepolypeptides from a single ORF.

Accordingly, the term “self-cleavage site” as used herein refers to acleavage site within the amino acid or nucleotide sequence where thissequence is cleaved or is cleavable without such cleavage involving anyadditional molecule or where the peptide- or phosphodiester-bondformation in this sequence is prevented in the first place (e.g. throughco-translational self-processing as described above).

It is understood that cleavage sites typically comprise several aminoacids or are encoded by several codons (e.g. in those cases, wherein the“cleavage site” is not translated into protein but leads to aninterruption of translation). Thus, the cleavage site may also serve thepurpose of a peptide linker, i.e. sterically separates two peptides.Thus, in some embodiments a “cleavage site” is both a peptide linker andprovides above described cleavage function. In this embodiment thecleavage site may encompass additional N- and/or C-terminal amino acids.

The term “host cell” as used herein refers to a cell that harbours avector (e.g. a plasmid or virus). Such host cell may either be aprokaryotic (e.g. a bacterial cell) or a eukaryotic cell (e.g. a fungal,plant or animal cell).

“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

The term “carrier”, as used herein, refers to a pharmacologicallyinactive substance such as but not limited to a diluent, excipient, orvehicle with which the therapeutically active ingredient isadministered. Such pharmaceutical carriers can be liquid or solid.Liquid carrier include but are not limited to sterile liquids, such assaline solutions in water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. A saline solution is a preferredcarrier when the pharmaceutical composition is administeredintravenously or intranasally by a nebulizer.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like.

Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

The term “composition” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus, in association with it.

The term “adjuvant” refers to agents that augment, stimulate, activate,potentiate, or modulate the immune response to the active ingredient ofthe composition at either the cellular or humoral level, e.g.immunologic adjuvants stimulate the response of the immune system to theactual antigen, but have no immunological effect themselves. Examples ofsuch adjuvants include but are not limited to inorganic adjuvants (e.g.inorganic metal salts such as aluminium phosphate or aluminiumhydroxide), organic adjuvants (e.g. saponins or squalene), oil-basedadjuvants (e.g. Freund's complete adjuvant and Freund's incompleteadjuvant), cytokines (e.g. IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, andINF-γ) particulate adjuvants (e.g. immuno-stimulatory complexes(ISCOMS), liposomes, or biodegradable microspheres), virosomes,bacterial adjuvants (e.g. monophosphoryl lipid A, or muramyl peptides),synthetic adjuvants (e.g. non-ionic block copolymers, muramyl peptideanalogues, or synthetic lipid A), or synthetic polynucleotides adjuvants(e.g polyarginine or polylysine).

The term “active ingredient” refers to the substance in a pharmaceuticalcomposition or formulation that is biologically active, i.e. thatprovides pharmaceutical value. A pharmaceutical composition may compriseone or more active ingredients which may act in conjunction with orindependently of each other.

The active ingredient can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as but not limited to those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, and the like.

As used herein, a “patient” means any mammal, reptile or bird that maybenefit from a treatment with a tumour vaccine described herein.Preferably, a “patient” is selected from the group consisting oflaboratory animals (e.g. mouse or rat), domestic animals (including e.g.guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken,camel, cat, dog, turtle, tortoise, snake, or lizard), or primatesincluding chimpanzees, bonobos, gorillas and human beings. It isparticularly preferred that the “patient” is a human being.

As used herein, “treat”, “treating” or “treatment” of a disease ordisorder means accomplishing one or more of the following: (a) reducingthe severity of the disorder; (b) limiting or preventing development ofsymptoms characteristic of the disorder(s) being treated; (c) inhibitingworsening of symptoms characteristic of the disorder(s) being treated;(d) limiting or preventing recurrence of the disorder(s) in patientsthat have previously had the disorder(s); and (e) limiting or preventingrecurrence of symptoms in patients that were previously symptomatic forthe disorder(s).

As used herein, “prevent”, “preventing”, “prevention”, or “prophylaxis”of a disease or disorder means preventing that such disease or disorderoccurs in patient.

As used herein, “administering” includes in vivo administration, as wellas administration directly to tissue ex vivo, such as vein grafts.

An “effective amount” is an amount of a therapeutic agent sufficient toachieve the intended purpose. The effective amount of a giventherapeutic agent will vary with factors such as the nature of theagent, the route of administration, the size and species of the animalto receive the therapeutic agent, and the purpose of the administration.The effective amount in each individual case may be determinedempirically by a skilled artisan according to established methods in theart.

EMBODIMENTS OF THE INVENTION

The present invention will now be further described. In the followingpassages different aspects of the invention are defined in more detail.Each aspect so defined may be combined with any other aspect or aspectsunless clearly indicated to the contrary. In particular, any featureindicated as being preferred or advantageous may be combined with anyother feature or features indicated as being preferred or advantageous.

In a first aspect the invention provides an expression system comprisinga first polynucleotide encoding at least one protein, peptide or variantthereof, which induces a T cell response and a second polynucleotideencoding at least one protein, which induces an anti-pathogenic B cellresponse. One of the advantages provided by the present invention is thefact that the B cell response to the protein, peptide or variant thereofinducing an antipathogenic B cell response can be enhanced, if at thesame time a protein inducing a T cell response is administered.

In the context of the present invention the term “expression system”preferably refers to one or more polynucleotide sequences comprising inaddition to the first and second polynucleotide the elements to directtranscription and translation of the proteins encoded by the first andsecond or any further polynucleotide, which may be included in thepreferred embodiments outlined below. Such elements included promoterand enhancer elements to direct transcription of mRNA in a cell-free ora cell-based based system, preferably a cell-based system. In anotherembodiment, wherein the polynucleotides are provided as translatableRNAs is envisioned that the expression system comprises those elementsthat are necessary for translation and/or stabilization of RNAs encodingthe T cell and B cell inducing protein, e.g. polyA-tail, IRES, capstructures etc.

According to a preferred embodiment of the first aspect, the firstpolynucleotide encodes a protein which induces a reaction of the immunesystem (i.e. immune response) in a host which is mediated by T cells. AT cell response involves the activation of antigen-specific T lymphocytesuch as but not limited to cytotoxic T cells (CTLs), T helper cells(T_(H) cells), central memory T cells (TCM cells), effector memory Tcells (TEM cells), and regulatory T cells (Treg cells). A T cellresponse against a protein is induced, if peptides of the protein areprocessed within the cell and presented to T cells on the surface of thecell via the MHC I or MHC II pathway. Thus, in the context of thepresent invention preferably those proteins or parts thereof are usedfor inducing a T cell response that are normally not exposed to, e.g.non structural or internal proteins or parts of structural or internalproteins of a virus not accessible to B-cells.

The second polynucleotide encodes a protein, peptide or variant thereofthat induces an anti-pathogenic B cell response. A B cell response is animmune response based on the activation of B lymphocytes, which produceand secrete antigen specific antibodies. B cells involved in such immuneresponse include but are not limited to plasma B cells, memory B cellsand B-1 cells. Thus, in the context of the present invention preferablythose pathogenic, e.g. viral, proteins or parts thereof are used forinducing a B cell response that are exposed on the outside of the virus,e.g. structural proteins or at least those parts of structural proteinsaccessible to B-cells on the outside of the pathogen (virus).

In embodiments of the first aspect of the present invention, the firstand the second polynucleotide are comprised on separate vectors or onthe same vector. Accordingly, the first polynucleotide may be comprisedon one vector and the second polynucleotide may be comprised on a secondvector. Alternatively or additionally, the first and the secondpolynucleotide may be comprised on the same vector. It is preferred thatthe first and the second polynucleotide are comprised on the samevector. It is particularly preferred that the first and the secondpolynucleotide comprised on the same vector are linked in such that theyare expressed as a polyprotein. Preferably, the first and the secondpolynucleotide form an open reading frame.

It is preferred that the first and the second polynucleotide areexpressed as an artificial polyprotein. In the context of the presentinvention the term “artificial polyprotein” is directed at polyproteinswhich are not naturally occurring, e.g. which are generated by usingrecombinant DNA techniques. Accordingly, the proteins, peptides orvariants thereof encoded in this artificial polyprotein are preferablyderived from pathogens which genome do not encode a polyproteincomprising the proteins, peptides or variants encoded by the first andsecond polynucleotide (and, optionally, the third polynucleotide) of theinvention. Preferably, the first and second polynucleotides are derivedfrom viruses, encoding no polyprotein or a polyprotein wherein therespective polynucleotides have a different order and/or sequence. Morepreferably, the first and second polynucleotide are derived from a viruswhich is selected from the group consisting of a DNA virus, a negativesense single stranded (ssRNA(−)) RNA virus or an ambisense RNA virus.Further preferred, the virus is selected from negative-single stranded(ssRNA(−)) RNA virus. Even more preferred, the virus is selected fromenveloped ssRNA(−) viruses, more preferably from the group consisting ofparamyxoviruses and orthomyxoviruses.

In preferred embodiments of the first aspect the protein, which inducesa T cell response is a non-structural and/or internal protein of avirus, and/or the protein, which induces an anti-pathogenic B cellresponse is a structural and/or surface protein of a pathogen,preferably a virus, wherein the virus is preferably selected from thegroup consisting of a DNA virus, a negative-strand RNA virus or anambisense RNA virus. Even more preferred, the virus is selected fromnegative-single stranded (ssRNA(−)) RNA virus. Even more preferred, thevirus is selected from enveloped ssRNA(−) viruses, more preferably fromthe group consisting of paramyxoviruses and orthomyxoviruses.

It is preferred that the amino acid sequence of the structural (surface)and/or non-structural (internal) protein comprises consecutive segmentsor a consensus sequence of one or more different virus isolates.

In the context of the present invention it is preferred that the term“segment” refers to a part of a protein or polyprotein. It isparticularly preferred that such segment folds and/or functionsindependently of the rest of the protein or polyprotein such as but notlimited to a domain, an epitope or a fragment thereof. It is understoodthat a protein variant in the context of the present invention differsin comparison to its parent polypeptide in changes in the amino acidsequence such as amino acid exchanges, insertions, deletions, N-terminaltruncations, or C-terminal truncations, or any combination of thesechanges, which may occur at one or several sites whereby the variantexhibits at least 80% sequence identity to its parent polypeptide.

In preferred embodiments, the structural protein, peptide or a variantthereof is a protein or peptide exposed on the surface of the nativepathogen, e.g. a virus. It is preferred that the structural and/orsurface protein triggers a T-cell independent immune response such asbut not limited to an antibody mediated immune response or an activationof the complement system. In a particularly preferred embodiment, thestructural and/or surface protein induces an antibody mediated immuneresponse. Such antibody mediated immune response is based on theactivation of B cells which produce and secrete antigen specificantibodies. B cells involved in such immune response include but are notlimited to plasma B cells, memory B cells and B-1 cells.

According to a preferred embodiment of the first aspect, the secondpolynucleotide encodes a protein or variant thereof that induces ananti-pathogenic B cell response. A B cell response is an immune responsebased on the activation of B lymphocytes, which produce and secreteantigen specific antibodies. B cells involved in such immune responseinclude but are not limited to plasma B cells, memory B cells and B-1cells. Thus, in the context of the present invention preferably thoseproteins or parts thereof are used for inducing a B cell response thatare exposed on the outside of a virus, e.g. structural and/or surfaceproteins or at least those parts of structural and/or surface proteinsaccessible to B-cells on the outside of a virus. An anti-pathogenic Bcell response is a B cell response directed against a pathogen whichinactivates, eliminates, blocks and/or neutralizes the respectivepathogen such that the disease caused by the pathogen does not break outand/or the symptoms are alleviated. In preferred embodiments of theinvention, the anti-pathogenic B cell response is effected by antibodiesthat bind to the surface of a pathogenic organism and attract the firstcomponent of the complement cascade with their Fc region and initiateactivation of the “classical” complement system. This results inpathogen elimination by two mechanisms. First, the binding of theantibody and complement molecules marks the pathogen for ingestion byphagocytes in a process called opsonization. Secondly, some complementsystem components form a membrane attack complex to assist antibodies todestroy the pathogen directly. Alternatively, the anti-pathogenic B cellresponse is effected by antibodies that bind to the pathogen'sstructural proteins blocking the attachment to cellular receptors. Inthis way, the antibody can neutralize the infection. As a furtheralternative, the anti-pathogenic B cell response is effected byantibodies that bind at a specialized region of the pathogen's surfaceprotein, the fusion peptide, which is necessary for the entry of thepathogen into the host cell. The antibody binding results in fixing theprotein in a pre-fusion state and blocking infection. The ability of aprotein or variant to induce B cell response which is anti-pathogeniccan be determined by the skilled person by applying tests and/or assayswell known in the art.

In a further preferred embodiment, a membrane attachment domain of theprotein exposed on the surface of the native virus or variant thereof isfunctionally deleted, thus, either being structurally deleted orstructurally present but not fulfilling its biological function. In aparticularly preferred embodiment, the amino acid sequence correspondingto the membrane attachment domain is deleted. The deletion of themembrane attachment region serves the purpose of ascertaining that theanti-pathogenic B cell response inducing protein is secreted from thecell into which the expression system of the invention has beenintroduced.

In a further preferred embodiment the anti-pathogenic B cell responseinducing protein comprises a secretion signal, which targets the proteinto the endoplasmatic reticulum (ER). Such secretion signals are presentpreferably in the context of a deleted membrane attachment domain. Theskilled person is well aware of various such secretion signals, whichmay be used as heterologous secretion signals, e.g. added to theN-terminus of the anti-pathogenic B cell response inducing protein.Alternatively or additionally a naturally occurring secretion signal maybe used, which is, e.g., present in the majority of structural and/orsurface viral proteins. Thus, if naturally present in the respectiveprotein it is preferred that the secretion signal is maintained in amodified version of the structural and/or surface protein.

In embodiments of the first aspect, the non-structural protein is aconserved internal protein suitable for inducing a T cell mediatedimmune response against the pathogen, preferably the viruses, involvingthe activation of antigen-specific T lymphocyte such as but not limitedto cytotoxic T cells (CTLs), T helper cells (T_(H) cells), centralmemory T cells (TCM cells), effector memory T cells (TEM cells), andregulatory T cells (Treg cells). Thus, preferably the T cell inducingprotein of the pathogen (virus) does not comprise a secretion signal.

In the context of the present invention, the protein, peptide or variantthereof encoded by the first polynucleotide is located either N- orC-terminally with respect to the protein, peptide or variant thereofencoded by the second polynucleotide. In a preferred embodiment, theprotein, peptide or variant thereof encoded by the second polynucleotideis located C-terminally with respect to the protein, peptide or variantthereof encoded by the first polynucleotide.

Accordingly, embodiments of the present invention have the formula X-Yor Y-X, wherein “X” depicts the T cell response inducing protein and “Y”depicts the anti-pathogenic B cell response inducing protein and a“dash” depicts a peptide bond.

In preferred embodiments of the first aspect, a polynucleotide encodinga cleavage site is positioned between the first polynucleotide and thesecond polynucleotide. It is within the scope of the present inventionthat every protein can be combined with any other protein and that anytwo proteins can or cannot be connected or linked by a cleavage site.

It is preferred that this cleavage site is either a self-cleaving site(i.e. a cleavage site within the amino acid sequence where this sequenceis cleaved or is cleavable without such cleavage involving anyadditional molecule or where the peptide-bond formation in this sequenceis prevented in the first place) or an endopeptidase cleavage site (i.e.a cleavage cite within the amino acid sequence where this sequence iscleaved or is cleavable by an endopeptidase, e.g. trypsin, pepsin,elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8,cathepsins). More preferably, the self-cleaving site is a 2A cleavagesite selected from the group consisting of a viral 2A peptide or 2A-likepeptide of Picornavirus, insect viruses, Aphtoviridae, Rotaviruses andTrypanosoma, preferably wherein the 2A cleavage site is the 2 A peptideof foot and mouth disease virus. Alternatively or additionally, thepolyprotein of the present invention can be cleaved by an autoprotease,i.e. a protease which cleaves peptide bonds in the same protein moleculewhich also comprises the protease. Examples of such autoproteases arethe NS2 protease from flaviviruses or the VP4 protease of birnaviruses

In the context of the present invention, the cleavage site can bepositioned N-terminally with respect to the protein, peptide or variantthereof encoded by the first polynucleotide and C-terminally withrespect to the protein, peptide or variant thereof encoded by the secondpolynucleotide. Alternatively the cleavage site can be positionedC-terminally with respect to the protein, peptide or variant thereofencoded by the first polynucleotide and N-terminally with respect to theprotein, peptide or variant thereof encoded by the secondpolynucleotide.

Accordingly, embodiments of the present invention have the formula X-C-Yor Y-C-X, wherein X” depicts the T cell response inducing protein and“Y” depicts the anti-pathogenic B cell response inducing protein, “C”depicts a cleavage site, and a “dash” depicts a peptide bond.

In a preferred embodiment of the first aspect, the expression systemfurther comprises a third polynucleotide encoding a protein, peptide ora variant thereof of a pathogen.

It is preferred that the protein, peptide or variant thereof encoded bythe third polynucleotide is a protein, peptide or variant thereofinducing a T cell response, preferably the third polynucleotide is aprotein, peptide or variant thereof which is a non-structural orinternal protein, peptide or variant thereof inducing a T cell response.

It is preferred that the protein, peptide or variant thereof encoded bythe third polynucleotide differs from the protein, peptide or variantthereof encoded by the first polynucleotide or the secondpolynucleotide. Preferably, the proteins, peptides or variants thereofencoded by the first, second and the third polynucleotide differ fromeach other in that they comprise amino acid sequences of differentproteins.

In preferred embodiments a polynucleotide encoding a linker ispositioned between the second polynucleotide and the thirdpolynucleotide. It is preferred that the linker is a flexible linker,preferably a flexible linker comprising an amino acid sequence accordingto SEQ ID NO: 6 (Gly-Gly-Gly-Ser-Gly-Gly-Gly).

In preferred embodiments the third polynucleotide is comprised on aseparate or on the same vector as the first polynucleotide and/or thesecond polynucleotide.

Accordingly, the first polynucleotide is comprised on one vector and thesecond polynucleotide is comprised on a second vector and the thirdpolynucleotide is comprised on a third vector. Alternatively oradditionally, the first and the second polynucleotide are comprised onthe same vector and the third polynucleotide is comprised on a separatevector, or the first and the third polynucleotide are comprised on thesame vector and the second polynucleotide is comprised on a separatevector, or the second and the third polynucleotide are comprised on thesame vector and the first polynucleotide is comprised on a separatevector. Alternatively or additionally, the first and the second and thethird polynucleotide are comprised on the same vector. It is preferredthat the first and the second and the third polynucleotide may becomprised on the same vector. It is particularly preferred that thefirst and the second and the third polynucleotide comprised on the samevector are linked in such that they are expressed as a polyprotein.Preferably, the first and the second and the third polynucleotidecomprised on the same vector form an open reading frame and, preferably,are expressed as a polyprotein.

In preferred embodiments of this aspect, the protein encoded by thesecond polynucleotide is located N-terminally with respect to theprotein encoded by the first polynucleotide and/or the protein of theoptional third polynucleotide, or the protein encoded by the secondpolynucleotide is located C-terminally with respect to the proteinencoded by the first polynucleotide and/or the protein of the optionalthird polynucleotide.

In even more preferred embodiments of this aspect, the firstpolynucleotide is located N-terminally with respect to the proteinencoded by the second polynucleotide and/or the protein of the optionalthird polynucleotide is located N-terminally with respect to the proteinencoded by the first polynucleotide or is located between the proteinencoded by the second polynucleotide and the protein encoded by thefirst polynucleotide; or the protein encoded by the first polynucleotideis located C-terminally with respect to the protein encoded by thesecond polynucleotide and/or the protein of the optional thirdpolynucleotide is located C-terminally with respect to the proteinencoded by the first polynucleotide or is located between the proteinencoded by the second polynucleotide and the protein encoded by thefirst polynucleotide.

Preferred embodiments of the present invention have the formula X-K-Y,Y-K-X, X-K-Y-Y, Y-Y-K-X, X-Y-K-Y, Y-K-Y-X, X-K-Y-K-Y, Y-K-Y-K-X, X-C-Y,Y-C-X, X-C-Y-Y, Y-Y-C-X, X-Y-C-Y, Y-C-Y-X, X-C-Y-C-Y, Y-C-Y-C-X,X-K-Y-C-Y, Y-C-Y-K-X, X-C-Y-K-Y, or Y-K-Y-C-X, wherein “X” depicts thesecond polynucleotide encoding at least one protein, peptide or variantthereof, which induces an antipathogenic B cell response and “Y” depictsthe first polynucleotide encoding at least one protein, peptide orvariant thereof, which induces an T cell response, “K” indicates thatone or more peptide linkers are present in this position, “C” indicatesthat one or more cleavage sites are present in this position and a“dash” depicts a peptide bond. Preferred arrangements are Y-K-Y-C-X orX-C-Y-K-Y.

It is further preferred that the non-structural and/or internal proteinencoded by the third polynucleotide is a conserved internal proteinsuitable for inducing a T cell mediated immune response against thevirus involving the activation of antigen-specific T lymphocyte such asbut not limited to cytotoxic T cells (CTLs), T helper cells (T_(H)cells), central memory T cells (TCM cells), effector memory T cells (TEMcells), and regulatory T cells (Treg cells).

It is within the scope of the present invention that every protein canbe combined with any other protein and that any two proteins can orcannot be connected or linked by either a cleavage site or a linkerpeptide.

In preferred embodiments, the vector or vectors comprising the first,and the second and/or the third polynucleotide is/are selected from thegroup consisting of plasmid, cosmid, phage, virus, and artificialchromosome. More preferably, a vector suitable for practicing thepresent invention is selected from the group consisting of plasmidvectors, cosmid vectors, phage vectors, preferably lambda phage andfilamentous phage vectors, viral vectors, adenovirus vectors (e.g.,non-replicating Ad5, Ad11, Ad26, Ad35, Ad49, ChAd3, ChAd4, ChAd5, ChAd6,ChAd7, ChAd8, ChAd9, ChAd10, ChAd1, ChAd16, ChAd17, ChAd19, ChAd20,ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd55,ChAd63, ChAd 73, ChAd82, ChAd83, ChAd146, ChAd147, PanAd1, PanAd2, andPanAd3 vectors or replication-competent Ad4 and Ad7 vectors),adeno-associated virus (AAV) vectors (e.g., AAV type 5 and type 2),alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE),sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN chimeras),herpes virus vectors (e.g. vectors derived from cytomegaloviruses, likerhesus cytomegalovirus (RhCMV) (14)), arena virus vectors (e.g.lymphocytic choriomeningitis virus (LCMV) vectors (15)), measles virusvectors, pox virus vectors (e.g., vaccinia virus, modified vacciniavirus Ankara (MVA), NYVAC (derived from the Copenhagen strain ofvaccinia), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV)vectors), vesicular stomatitis virus vectors, retrovirus, lentivirus,viral like particles, and bacterial spores. The vectors ChAd3, ChAd4,ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17,ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38,ChAd44, ChAd63 and ChAd82 are described in detail in WO 2005/071093. Thevectors PanAd1, PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAd146, andChAd147 are described in detail in WO 2010/086189. It is particularlypreferred that the vector is selected from the group consisting of MVA,ChAd63 and PanAd3.

In preferred embodiments, the expression system is for use in medicine.In more preferred embodiments, the expression system is for use in theprophylaxis or treatment of an infection and/or in the manufacturing ofmedicament for use in the prophylaxis or treatment of an infectionand/or for use in methods of prophylaxis or treatment of an infection,wherein the infection is preferably a viral infection, particularlypreferably for use in the prophylaxis or treatment of a pathogen and/orin the manufacturing of medicament for use in the prophylaxis ortreatment of a pathogen and/or for use in methods of prophylaxis ortreatment of a pathogen, wherein the pathogen preferably is a virus.Preferably, the expression system is for use in the prophylaxis ortreatment of an infection by a virus and/or in the manufacturing ofmedicament for use in the prophylaxis or treatment of a virus and/or foruse in methods of prophylaxis or treatment of a virus, wherein thepathogen selected from the group of a DNA virus, a negative-singlestranded (ssRNA(−)) RNA virus or an ambisense RNA virus. Furtherpreferred the expression system is for use in the prophylaxis ortreatment and/or in the manufacturing of medicament for use in theprophylaxis or treatment of a virus and/or for use in methods ofprophylaxis or treatment of an infection by negative sensesingle-stranded (ssRNA(−)) RNA virus. Even more preferred, theexpression system is for use in the prophylaxis or treatment and/or inthe manufacturing of medicament for use in the prophylaxis or treatmentof a virus and/or for use in methods of prophylaxis or treatment of aninfection by a virus selected from enveloped ssRNA(−) viruses, morepreferably from the group consisting of paramyxoviruses andorthomyxoviruses.

In preferred embodiments the expression system is for use in enhancingan immune response. In more preferred embodiments, the expression systemis for use in enhancing a B cell immune response against an immunogen,preferably a pathogen, more preferably a virus as defined above.

According to a preferred embodiment of the first aspect, the firstpolynucleotide encodes a viral protein of a paramyxovirus or variantthereof which induces a reaction of the immune system (i.e. immuneresponse) in a host which is mediated by T cells, and the secondpolynucleotide encodes a viral protein of a paramyxovirus or variantthereof that induces an anti-pathogenic B cell response againstparamyxoviruses. It is preferred that the paramyxovirus whose viralproteins are encoded for by the first and second polynucleotide isselected from the subfamily of Pneumovirinae, Paramyxovirinae,Fer-de-Lance-Virus, Nariva-Virus, Salem-Virus, Tupaia-Paramyxovirus,Beilong-Virus, J-Virus, Menangle-Virus, Mossmann-Virus, andMurayama-Virus. In even more preferred embodiments, the Pneumovirinae isselected from the group consisting of Pneumovirus, preferably humanrespiratory syncytial virus (RSV), murine pneumonia virus, bovine RSV,ovine RSV, caprine RSV, turkey rinotracheitis virus, andMetapneumovirus, preferably human metapneumovirus (hMPV) and avianmetapneumovirus. In even more preferred embodiments, the Paramyxovirinaeis selected from the group consisting of Respirovirus, preferably humanparainfluenza virus 1 and 3, and Rubulavirus, preferably humanparainfluenza virus 2 and 4.

In embodiments of the first aspect of the present invention, the firstand the second polynucleotide are comprised on separate vectors or onthe same vector. Accordingly, the first polynucleotide may be comprisedon one vector and the second polynucleotide may be comprised on a secondvector. Alternatively or additionally, the first and the secondpolynucleotide may be comprised on the same vector. It is preferred thatthe first and the second polynucleotide are comprised on the samevector. It is particularly preferred that the first and the secondpolynucleotide comprised on the same vector are linked in such that theyare expressed as a polyprotein. Preferably, the first and the secondpolynucleotide form an open reading frame.

According to preferred embodiments of the first aspect the firstpolynucleotide encodes a viral protein of a paramyxovirus or variantthereof which induces a reaction of the immune system (i.e. immuneresponse) in a host which is mediated by T cells. A T cell responseinvolves the activation of antigen-specific T lymphocyte such as but notlimited to cytotoxic T cells (CTLs), T helper cells (T_(H) cells),central memory T cells (TCM cells), effector memory T cells (TEM cells),and regulatory T cells (Treg cells). A T cell response against a proteinis induced, if peptides of the protein are processed within the cell andpresented to T cells on the surface of the cell via the MHC I or MHC IIpathway. Thus, in the context of the present invention preferably thoseviral proteins or parts thereof are used for inducing a T cell responsethat are normally not exposed on the outside of the virus, e.g. nonstructural or internal proteins or parts of structural or surfaceproteins not accessible to B-cells on the outside of the virus.

The second polynucleotide encodes a viral protein of a paramyxovirus orvariant thereof that induces an anti-pathogenic B cell response againstthe paramyxovirus. A B cell response is an immune response based on theactivation of B lymphocytes, which produce and secrete antigen specificantibodies. B cells involved in such immune response include but are notlimited to plasma B cells, memory B cells and B-1 cells. Thus, in thecontext of the present invention preferably those viral proteins orparts thereof are used for inducing an anti-pathogenic B cell responsethat are exposed on the outside of the virus, e.g. structural proteinsor at least those parts of structural proteins accessible to B-cells onthe outside of the virus.

According to a preferred embodiment of the first aspect, the secondpolynucleotide encodes a viral protein of a paramyxovirus or variantthereof that induces an anti-pathogenic B cell response. A B cellresponse is an immune response based on the activation of B lymphocytes,which produce and secrete antigen specific antibodies. B cells involvedin such immune response include but are not limited to plasma B cells,memory B cells and B-1 cells. Thus, in the context of the presentinvention preferably those viral proteins or parts thereof are used forinducing an anti-pathogenic B cell response that are exposed on theoutside of the virus, e.g. structural and/or surface proteins or atleast those parts of structural and/or surface proteins accessible toB-cells on the outside of the virus.

In preferred embodiments of the first aspect the viral protein of aparamyxovirus, which induces a T cell response is a non-structuraland/or internal protein of a paramyxovirus, and/or the viral protein ofa paramyxovirus, which induces an anti-pathogenic B cell response is astructural and/or surface protein of a paramyxovirus.

It is preferred that the amino acid sequence of the structural (surface)and/or non-structural (internal) protein comprises consecutive segmentsor a consensus sequence of one or more different paramyxovirus isolates.

In preferred embodiments, the structural protein is a protein exposed onthe surface of the native paramyxovirus or a variant thereof. It ispreferred that the structural protein triggers a T-cell independentimmune response such as but not limited to an antibody mediated immuneresponse or an activation of the complement system. In a particularlypreferred embodiment, the structural and/or surface protein induces anantibody mediated immune response. Such antibody mediated immuneresponse is based on the activation of B cells which produce and secreteantigen specific antibodies. B cells involved in such immune responseinclude but are not limited to plasma B cells, memory B cells and B-1cells.

In a further preferred embodiment, the membrane attachment domain of theprotein exposed on the surface of the native paramyxovirus or variantthereof is functionally deleted, thus, either being structurally deletedor structurally present but not fulfilling its biological function. In aparticularly preferred embodiment, the amino acid sequence correspondingto the membrane attachment domain is deleted. The deletion of themembrane attachment region serves the purpose of ascertaining that theanti-pathogenic B cell response inducing protein is secreted from thecell into which the expression system of the invention has beenintroduced.

In a further preferred embodiment the anti-pathogenic B cell responseinducing paramyxovirus protein comprises a secretion signal, whichtargets the protein to the endoplasmatic reticulum (ER). Thus, ifnaturally present in the respective structural or surface protein it ispreferred that the secretion signal is maintained in a modified versionof the structural or surface protein.

It is further preferred that the structural and/or surface protein ofthe native paramyxovirus is selected from the group consisting of fusionprotein (F) and any of the attachment glycoproteins G, H, and HN.

The attachment glycoproteins are found in all enveloped viruses andmediate the initial interaction between the viral envelope and theplasma membrane of the host cell via their binding to carbohydratemoieties or cell adhesion domains of proteins or other molecules on theplasma membrane of the host cell. Thereby, attachment glycoproteinsbridge the gap between the virus and the membrane of the host cell.Attachment glycoproteins designated as “H” possess hemagglutininactivity and are found in morbilliviruses and henipaviruses,glycoproteins designated as “HN possess hemagglutinin and neuraminidaseactivities and are found in respiroviruses, rubulaviruses andavulaviruses. Attachment glycoproteins are designated as “G” when theyhave neither haemagglutination nor neuraminidase activity. G attachmentglycoproteins can be found in all members of Pneumovirinae.

Fusion protein “F” is found in all enveloped viruses and mediates thefusion of the viral envelope with the plasma membrane of the host cell.F is a type I glycoprotein that recognizes receptors present on the cellsurface of the host cell to which it binds. F consists of a fusionpeptide adjacent to which the transmembrane domains are located,followed by two heptad repeat (HR) regions, HR1 and HR2, respectively.Upon insertion of the fusion peptide into the plasma membrane of thehost cell, the HR1 region forms a trimeric coiled coil structure intowhose hydrophobic grooves the HR2 regions folds back. Thereby, a hairpinstructure is formed that draws the viral lipid bilayer and cellularplasma membrane even closer together and allows for the formation of afusion pore and consecutively the complete fusion of both lipid bilayersenabling the virus capsid to enter into the cytoplasm of the host cell.All of these features are common in fusion-mediating proteins ofenveloped viruses.

In a preferred embodiment of the first aspect, F comprises, essentiallyconsists of or consists of an amino acid sequence of F of one RSVisolate or a consensus amino acid sequence of two or more different RSVisolates, preferably according to SEQ ID NO: 1, more preferablyaccording to SEQ ID NO: 2 or a variant thereof.

In preferred embodiments of the first aspect, the non-structural proteinis a conserved internal protein of paramyxoviruses suitable for inducinga T cell mediated immune response against the paramyxovirus, involvingthe activation of antigen-specific T lymphocyte such as but not limitedto cytotoxic T cells (CTLs), T helper cells (T_(H) cells), centralmemory T cells (TCM cells), effector memory T cells (TEM cells), andregulatory T cells (Treg cells). Thus, preferably the T cell inducingprotein of the paramyxovirus does not comprise a secretion signal.

Preferably, the non-structural and/or internal protein is selected fromthe group consisting of nucleoprotein N, Matrix proteins M and M2,Phosphoprotein P, non structural proteins NS1 and NS2, and the catalyticsubunit of the polymerase (L).

The nucleoprotein N serves several functions which include theencapsidation of the RNA genome into a RNAase-resistant nucleocapsid. Nalso interacts with the M protein during virus assembly and interactswith the P-L polymerase during transcription and replication of thegenome.

The matrix protein M is the most abundant protein in paramyxovirus andis considered to be the central organizer of viral morphology byinteracting with the cytoplasmatic tail of the integral membraneproteins and the nucleocapsid. M2 is a second membrane-associatedprotein that is not glycosylated and is mainly found in pneumovirus.

Phosphoprotein P binds to the N and L proteins and forms part of the RNApolymerase complex in all paramyxoviruses. Large protein L is thecatalytic subunit of RNA-dependent RNA polymerase.

The function of non-structural proteins NS1 and NS2 has not yet beenidentified; however, there are indications that they are involved in theviral replication cycle.

In preferred embodiments, N comprises an amino acid sequence of N, ofone RSV isolate or a consensus amino acid sequence of two or moredifferent RSV isolates, preferably according to SEQ ID NO: 3 and whereinM2 comprises an amino acid sequence of M2 of one RSV isolate or aconsensus amino acid sequence of two or more different RSV isolates,preferably according to SEQ ID NO: 5. It is further preferred thatwherein N comprises the amino acid sequence according to SEQ ID NO: 4and M2 comprises the amino acid sequence according to SEQ ID NO: 5.

In the context of the present invention, the structural and/or surfaceprotein encoded by the first polynucleotide is located either N- orC-terminally with respect to the non-structural and/or internal proteinencoded by the second polynucleotide. In a preferred embodiment, thenon-structural and/or internal protein encoded by the secondpolynucleotide is located C-terminally with respect to the structuraland/or surface protein encoded by the first polynucleotide.

More specifically, N, M, M2, P, NS1, NS2, or L can be located N- orC-terminally of F, G, H, or HN. Preferably, N, M, M2, P, NS1, NS2, or Lare located C-terminally of F, G, H, or HN. In a more preferredembodiment N or M2 are located C-terminally of F. In a particularlypreferred embodiment N is located C-terminally of F.

Accordingly, embodiments of the present invention have the formula X-Yor Y-X, wherein “X” depicts F, G, H, or HN and “Y” depicts N, M, M2, P,NS1, NS2, or L and a “dash” depicts a peptide bond. Preferredarrangements are the following:

F-N, G-N, H-N, HN-N, F-M, G-M, H-M, HN-M, F-M2, G-M2, H-M2, HN-M2, F-P,G-P, H-P, HN-P, F-NS1, G-NS1, H-NS1, HN-NS1, F-NS2, G-NS2, H-NS2,HN-NS2, F-L, G-L, H-L, HN-L, N-F, N-G, N-H, N-HN, M-F, M-G, M-H, M-HN,M2-F, M2-G, M2-H, M2-HN, P-F, P-G, P-H, P-HN, NS1-FF, NS1-G, NS1-H,NS1-HN, NS2-F, NS2-G, NS2-H, NS2-HN, L-F, L-G, L-H, or L-HN.

It is within the scope of the present invention that every protein canbe combined with any other protein.

In preferred embodiments of the first aspect, a polynucleotide encodinga cleavage site is positioned between the first polynucleotide and thesecond polynucleotide.

It is preferred that this cleavage site is either a self-cleaving site(i.e. a cleavage site within the amino acid sequence where this sequenceis cleaved or is cleavable without such cleavage involving anyadditional molecule or where the peptide-bond formation in this sequenceis prevented in the first place) or an endopeptidase cleavage site (i.e.a cleavage cite within the amino acid sequence where this sequence iscleaved or is cleavable by an endopeptidase, e.g. trypsin, pepsin,elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8,cathepsins). More preferably, the self-cleaving site is a 2A cleavagesite selected from the group consisting of a viral 2A peptide or 2A-likepeptide of Picornavirus, insect viruses, Aphtoviridae, Rotaviruses andTrypanosoma, preferably wherein the 2A cleavage site is the 2 A peptideof foot and mouth disease virus.

In the context of the present invention, the cleavage site can bepositioned N-terminally with respect to the structural and/or surfaceprotein encoded by the first polynucleotide and C-terminally withrespect to the non-structural and/or internal protein encoded by thesecond polynucleotide. Alternatively the cleavage site can be positionedC-terminally with respect to the structural and/or surface proteinencoded by the first polynucleotide and N-terminally with respect to thenon-structural and/or internal protein encoded by the secondpolynucleotide. More specifically, the cleavage site can be positionedC- or N-terminally with respect to F, G, H, or HN and C- or N-terminallywith respect to N, M, M2, P, NS1, NS2, or L. In a preferred embodimentthe cleavage site is located N-terminally with respect to N, M, M2, P,NS1, NS2, or L and C-terminally with respect to F, G, H, or HN. It isparticularly preferred that the cleavage site is located N-terminallywith respect to N and C-terminally with respect to F.

Accordingly, embodiments of the present invention have the formula X-C-Yor Y-C-X, wherein “X” depicts F, G, H, or HN and “Y” depicts N, M, M2,P, NS1, NS2, or L, “C” depicts a cleavage site, and a “dash” depicts apeptide bond. Preferred arrangements are the following:

F-C-N, G-C-N, H-C-N, HN-C-N, F-C-M, G-C-M, H-C-M, HN-C-M, F-C-M2,G-C-M2, H-C-M2, HN-C-M2, F-C-P, G-C-P, H-C-P, HN-C-P, F-C-NS1, G-C-NS1,H-C-NS1, HN-C-NS1, F-C-NS2, G-C-NS2, H-C-NS2, HN-C-NS2, F-C-L, G-C-L,H-C-L, HN-C-L, N-C-F, N-C-G, N-C-H, N-C-HN, M-C-F, M-C-G, M-C-H, M-C-HN,M2-C-F, M2-C-G, M2-C-H, M2-C-HN, P-C-F, P-C-G, P-C-H, P-C-HN, NS1-C-FF,NS1-C-G, NS1-C-H, NS1-C-HN, NS2-C-F, NS2-C-G, NS2-C-H, NS2-C-HN, L-C-F,L-C-G, L-C-H, or L-C-HN. Particularly, preferred is F-C-N.

It is within the scope of the present invention that every protein canbe combined with any other protein and that any two proteins can orcannot be connected or linked by a cleavage site.

In preferred embodiment of the first aspect, the expression systemfurther comprises a third polynucleotide encoding a non-structuraland/or internal protein of a paramyxovirus or a variant thereof.Preferably, the non-structural and/or internal protein is of aparamyxovirus selected from the group consisting of Pneumovirinae,Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus, Salem-Virus,Tupaia-Paramyxovirus, Beilong-Virus, J-Virus, Menangle-Virus,Mossmann-Virus, and Murayama-Virus, more preferably, the Pneumovirinaeis selected from the group consisting of Pneumovirus, preferably humanrespiratory syncytical virus (RSV), murine pneumonia virus, bovine RSV,ovine RSV, caprine RSV, turkey rinotracheitis and Metapneumovirus,preferably human metapneumovirus, avaian metapneumovirus, morepreferably, the Paramyxovirinae is selected from the group consisting ofRespirovirus, preferably human parainfluenza virus 1 and 3, andRubulavirus, preferably human parainfluenza virus 2 and 4.

In preferred embodiments the third polynucleotide is comprised on aseparate or on the same vector as the first polynucleotide and/or thesecond polynucleotide.

Accordingly, the first polynucleotide is comprised on one vector and thesecond polynucleotide is comprised on a second vector and the thirdpolynucleotide is comprised on a third vector. Alternatively oradditionally, the first and the second polynucleotide are comprised onthe same vector and the third polynucleotide is comprised on a separatevector, or the first and the third polynucleotide are comprised on thesame vector and the second polynucleotide is comprised on a separatevector, or the second and the third polynucleotide are comprised on thesame vector and the first polynucleotide is comprised on a separatevector. Alternatively or additionally, the first and the second and thethird polynucleotide are comprised on the same vector. It is preferredthat the first and the second and the third polynucleotide may becomprised on the same vector. It is particularly preferred that thefirst and the second and the third polynucleotide comprised on the samevector are linked in such that they are expressed as a viralpolyprotein. Preferably, the first and the second and the thirdpolynucleotide comprised on the same vector form an open reading frame.

It is further preferred that the non-structural and/or internal proteinencoded by the third polynucleotide is a conserved internal proteinsuitable for inducing a T cell mediated immune response against thevirus involving the activation of antigen-specific T lymphocyte such asbut not limited to cytotoxic T cells (CTLs), T helper cells (T_(H)cells), central memory T cells (TCM cells), effector memory T cells (TEMcells), and regulatory T cells (Treg cells).

Preferably, the non-structural and/or internal protein is selected fromthe group consisting of nucleoprotein N, Matrix proteins M and M2,Phosphoprotein P, non structural proteins NS1 and NS2, and the catalyticsubunit of the polymerase (L).

In preferred embodiments, N comprises an amino acid sequence of N, ofone RSV isolate or a consensus amino acid sequence of two or moredifferent RSV isolates, preferably according to SEQ ID NO: 3 and whereinM2 comprises an amino acid sequence of M2 of one RSV isolate or aconsensus amino acid sequence of two or more different RSV isolates,preferably according to SEQ ID NO: 5. It is further preferred thatwherein N comprises the amino acid sequence according to SEQ ID NO: 4and M2 comprises the amino acid sequence according to SEQ ID NO: 5.

It is preferred that the non-structural and/or internal protein encodedby the third polynucleotide differs from the non-structural and/orinternal protein encoded by the second polynucleotide.

The non-structural and/or internal proteins encoded by the second andthe third polynucleotide differ from each other in that they compriseamino acid sequences of different viral proteins. For instance, thismeans that the non-structural and/or internal protein encoded by thesecond polynucleotide comprises the amino acid sequence of the N proteinwhilst the non-structural and/or internal protein encoded by the secondpolynucleotide comprises the amino acid sequence of the M2 protein orvice versa.

The non-structural and/or internal protein encoded by the thirdpolynucleotide can be located either N- or C-terminally of thenon-structural and/or internal protein encoded by the secondpolynucleotide. In a preferred embodiment of the first aspect, thenon-structural and/or internal protein encoded by the thirdpolynucleotide is located C-terminally of the non-structural and/orinternal protein encoded by the second polynucleotide. Morespecifically, N or M2 can be located N- or C-terminally of N or M2,preferably N is located C-terminally of M2.

In preferred embodiments a polynucleotide encoding a linker ispositioned between the second polynucleotide and the thirdpolynucleotide. It is preferred that the linker is a flexible linker,preferably a flexible linker comprising an amino acid sequence accordingto SEQ ID NO: 6.

In embodiments of the first aspect, the protein encoded by the secondpolynucleotide is located N-terminally with respect to the proteinencoded by the first polynucleotide and/or the protein of the optionalthird polynucleotide, or the protein encoded by the secondpolynucleotide is located C-terminally with respect to the proteinencoded by the first polynucleotide and/or the protein of the optionalthird polynucleotide.

In even more preferred embodiments of this aspect, the firstpolynucleotide is located N-terminally with respect to the proteinencoded by the second polynucleotide and/or the protein of the optionalthird polynucleotide is located N-terminally with respect to the proteinencoded by the first polynucleotide or is located between the proteinencoded by the second polynucleotide and the protein encoded by thefirst polynucleotide; or the protein encoded by the first polynucleotideis located C-terminally with respect to the protein encoded by thesecond polynucleotide and/or the protein of the optional thirdpolynucleotide is located C-terminally with respect to the proteinencoded by the first polynucleotide or is located between the proteinencoded by the second polynucleotide and the protein encoded by thefirst polynucleotide. More specifically, F, G, H, or HN are located C-or N-terminally with respect to N, M, M2, P, NS1, NS2, or L and N, M,M2, P, NS1, NS2, or L are located C- or N-terminally with respect to Nor M2. In a preferred embodiment F is located N-terminally with respectto N and M2 is located C-terminally with respect to N.

Accordingly, preferred embodiments of the present invention have theformula X-K-Y, Y-K-X, X-K-Y-Y, Y-Y-K-X, X-Y-K-Y, Y-K-Y-X, X-K-Y-K-Y,Y-K-Y-K-X, X-C-Y, Y-C-X, X-C-Y-Y, Y-Y-C-X, X-Y-C-Y, Y-C-Y-X, X-C-Y-C-Y,Y-C-Y-C-X, X-K-Y-C-Y, Y-C-Y-K-X, X-C-Y-K-Y, or Y-K-Y-C-X, wherein “X”depicts F, G, H, or HN and “Y” depicts N, M, M2, P, NS1, NS2, or L, “K”indicates that one or more peptide linkers are present in this position,“C” indicates that one or more cleavage sites are present in thisposition and a “dash” depicts a peptide bond. Preferred arrangements areX-C-Y-K-Y. Even more preferred arrangements are the following:

F-K-N, G-K-N, H-K-N, HN-K-N, F-K-M, G-K-M, H-K-M, HN-K-M, F-K-M2,G-K-M2, H-K-M2, HN-K-M2, F-K-P, G-K-P, H-K-P, HN-K-P, F-K-NS1, G-K-NS1,H-K-NS1, HN-K-NS1, F-K-NS2, G-K-NS2, H-K-NS2, HN-K-NS2, F-K-L, G-K-L,H-K-L, HN-K-L, N-K-F, N-K-G, N-K-H, N-K-HN, M-K-F, M-K-G, M-K-H, M-K-HN,M2-K-F, M2-K-G, M2-K-H, M2-K-HN, P-K-F, P-K-G, P-K-H, P-K-HN, NS1-K-FF,NS1-K-G, NS1-K-H, NS1-K-HN, NS2-K-F, NS2-K-G, NS2-K-H, NS2-K-HN, L-K-F,L-K-G, L-K-H, L-K-HN, F-C-N, G-C-N, H-C-N, HN-C-N, F-C-M, G-C-M, H-C-M,HN-C-M, F-C-M2, G-C-M2, H-C-M2, HN-C-M2, F-C-P, G-C-P, H-C-P, HN-C-P,F-C-NS1, G-C-NS1, H-C-NS1, HN-C-NS1, F-C-NS2, G-C-NS2, H-C-NS2,HN-C-NS2, F-C-L, G-C-L, H-C-L, HN-C-L, N-C-F, N-C-G, N-C-H, N-C-HN,M-C-F, M-C-G, M-C-H, M-C-HN, M2-C-F, M2-C-G, M2-C-H, M2-C-HN, P-C-F,P-C-G, P-C-H, P-C-HN, NS1-C-FF, NS1-C-G, NS1-C-H, NS1-C-HN, NS2-C-F,NS2-C-G, NS2-C-H, NS2-C-HN, L-C-F, L-C-G, L-C-H, or L-C-HN, F-N-M,G-N-M, H-N-M, HN-N-M, F-N-M2, G-N-M2, H-N-M2, HN-N-M2, F-N-P, G-N-P,H-N-P, HN-N-P, F-N-NS1, G-N-NS1, H-N-NS1, HN-N-NS1, F-N-NS2, G-N-NS2,H-N-NS2, HN-N-NS2, F-N-L, G-N-L, H-N-L, HN-N-L, F-M-N, G-M-N, H-M-N,HN-M-N, F-M-P, G-M-P, H-M-P, HN-M-P, F-M-NS1, G-M-NS1, H-M-NS1,HN-M-NS1, F-M-NS2, G-M-NS2, H-M-NS2, HN-M-NS2, F-M-L, G-M-L, H-M-L,HN-M-L, F-M2-N, G-M2-N, H-M2-N, HN-M2-N, F-M2-P, G-M2-P, H-M2-P,HN-M2-P, F-M2-NS1, G-M2-NS1, H-M2-NS1, HN-M2-NS1, F-M2-NS2, G-M2-NS2,H-M2-NS2, HN-M2-NS2, F-M2-L, G-M2-L, H-M2-L, HN-M2-L, F-P-N, G-P-N,H-P-N, HN-P-N, F-P-M, G-P-M, H-P-M, HN-P-M, F-P-M2, G-P-M2, H-P-M2,HN-P-M2, F-P-NS1, G-P-NS1, H-P-NS1, HN-P-NS1, F-P-NS2, G-P-NS2, H-P-NS2,HN-P-NS2, F-P-L, G-P-L, H-P-L, HN-P-L, F-NS1-N, G-NS1-N, H-NS1-N,HN-NS1-N, F-NS1-M, G-NS1-M, H-NS1-M, HN-NS1-M, F-NS1-M2, G-NS1-M2,H-NS1-M2, HN-NS1-M2, F-NS1-P, G-NS1-P, H-NS1-P, HN-NS1-P, F-NS1-NS2,G-NS1-NS2, H-NS1-NS2, HN-NS1-NS2, F-NS1-L, G-NS1-L, H-NS1-L, HN-NS1-L,F-NS2-N, G-NS2-N, H-NS2-N, HN-NS2-N, F-NS2-M, G-NS2-M, H-NS2-M,HN-NS2-M, F-NS2-M2, G-NS2-M2, H-NS2-M2, HN-NS2-M2, F-NS2-P, G-NS2-P,H-NS2-P, HN-NS2-P, F-NS2-NS1, G-NS2-NS1, H-NS2-NS1, HN-NS2-NS1, F-NS2-L,G-NS2-L, H-NS2-L, HN-NS2-L, F-L-N, G-L-N, H-L-N, HN-L-N, F-L-M2, G-L-M2,H-L-M2, HN-L-M2, F-L-P, G-L-P, H-L-P, HN-L-P, F-L-NS1, G-L-NS1, H-L-NS1,HN-L-NS1, F-L-NS2, G-L-NS2, H-L-NS2, HN-L-NS2, M-N-F, M-N-G, M-N-H,M-N-HN, M2-N-F, M2-N-G, M2-N-H, M2-N-HN, P-N-F, P-N-G, P-N-H, P-N-HN,NS1-N-F, NS1-N-G, NS1-N-H, NS1-N-HN, NS2-N-F, NS2-N-G, NS2-N-H,NS2-N-HN, L-N-F, L-N-G, L-N-H, L-N-HN, N-M-F, N-M-G, N-M-H, N-M-HN,P-M-F, P-M-G, P-M-H, P-M-HN, NS1-M-F, NS1-M-G, NS1-M-H, NS1-M-HN,NS2-M-F, NS2-M-G, NS2-M-H, NS2-M-HN, L-M-F, L-M-G, L-M-H, L-M-HN,N-M2-F, N-M2-G, N-M2-H, N-M2-HN, P-M2-F, P-M2-G, P-M2-H, P-M2-HN,NS1-M2-F, NS1-M2-G, NS1-M2-H, NS1-M2-HN, NS2-M2-F, NS2-M2-G, NS2-M2-H,NS2-M2-HN, L-M2-F, L-M2-G, L-M2-H, L-M2-HN, N-P-F, N-P-G, N-P-H, N-P-HN,M-P-F, M-P-G, M-P-H, M-P-HN, M2-P-F, M2-P-G, M2-P-H, M2-P-HN, NS1-P-F,NS1-P-G, NS1-P-H, NS1-P-HN, NS2-P-F, NS2-P-G, NS2-P-H, NS2-P-HN, L-P-F,L-P-G, L-P-H, L-P-HN, N-NS1-F, N-NS1-G, N-NS1-H, N-NS1-HN, M-NS1-F,M-NS1-G, M-NS1-H, M-NS1-HN, M2-NS1-F, M2-NS1-G, M2-NS1-H, M2-NS1-HN,P-NS1-F, P-NS1-G, P-NS1-H, P-NS1-HN, NS2-NS1-F, NS2-NS1-G, NS2-NS1-H,NS2-NS1-HN, L-NS1-F, L-NS1-G, L-NS1-H, L-NS1-HN, N-NS2-F, N-NS2-G,N-NS2-H, N-NS2-HN, M-NS2-F, M-NS2-G, M-NS2-H, M-NS2-HN, M2-NS2-F,M2-NS2-G, M2-NS2-H, M2-NS2-HN, P-NS2-F, P-NS2-G, P-NS2-H, P-NS2-HN,NS1-NS2-F, NS1-NS2-G, NS1-NS2-H, NS1-NS2-HN, L-NS2-F, L-NS2-G, L-NS2-H,L-NS2-HN, N-L-F, N-L-G, N-L-H, N-L-HN, M-L-F, M-L-G, M-L-H, M-L-HN,M2-L-F, M2-L-G, M2-L-H, M2-L-HN, P-L-F, P-L-G, P-L-H, P-L-HN, NS1-L-F,NS1-L-G, NS1-L-H, NS1-L-HN, NS2-L-F, NS2-L-G, NS2-L-H, NS2-L-HN,F-K-N-N, G-K-N-N, H-K-N-N, HN-K-N-N, F-K-N-M, G-K-N-M, H-K-N-M,HN-K-N-M, F-K-N-M2, G-K-N-M2, H-K-N-M2, HN-K-N-M2, F-K-N-P, G-K-N-P,H-K-N-P, HN-K-N-P, F-K-N-NS1, G-K-N-NS1, H-K-N-NS1, HN-K-N-NS1,F-K-N-NS2, G-K-N-NS2, H-K-N-NS2, HN-K-N-NS2, F-K-N-L, G-K-N-L, H-K-N-L,HN-K-N-L, F-C-N-N, G-C-N-N, H-C-N-N, HN-C-N-N, F-C-N-M, G-C-N-M,H-C-N-M, HN-C-N-M, F-C-N-M2, G-C-N-M2, H-C-N-M2, HN-C-N-M2, F-C-N-P,G-C-N-P, H-C-N-P, HN-C-N-P, F-C-N-NS1, G-C-N-NS1, H-C-N-NS1, HN-C-N-NS1,F-C-N-NS2, G-C-N-NS2, H-C-N-NS2, HN-C-N-NS2, F-C-N-L, G-C-N-L, H-C-N-L,HN-C-N-L, F-K-M-M, G-K-M-M, H-K-M-M, HN-K-M-M, F-K-M-M2, G-K-M-M2,H-K-M-M2, HN-K-M-M2, F-K-M-N, G-K-M-N, H-K-M-N, HN-K-M-N, F-K-M-P,G-K-M-P, H-K-M-P, HN-K-M-P, F-K-M-NS1, G-K-M-NS1, H-K-M-NS1, HN-K-M-NS1,F-K-M-NS2, G-K-M-NS2, H-K-M-NS2, HN-K-M-NS2, F-K-M-L, G-K-M-L, H-K-M-L,HN-K-M-L, F-K-M2-M, G-K-M2-M, H-K-M2-M, HN-K-M2-M, F-K-M2-N, G-K-M2-N,H-K-M2-N, HN-K-M2-N, F-K-M2-P, G-K-M2-P, H-K-M2-P, HN-K-M2-P,F-K-M2-NS1, G-K-M2-NS1, H-K-M2-NS1, HN-K-M2-NS1, F-K-M2-NS2, G-K-M2-NS2,H-K-M2-NS2, HN-K-M2-NS2, F-K-M2-L, G-K-M2-L, H-K-M2-L, HN-K-M2-L,F-K-P-N, G-K-P-N, H-K-P-N, HN-K-P-N, F-K-P-M, G-K-P-M, H-K-P-M,HN-K-P-M, F-K-P-M2, G-K-P-M2, H-K-P-M2, HN-K-P-M2, F-K-P-P, G-K-P-P,H-K-P-P, HN-K-P-P, F-K-P-NS1, G-K-P-NS1, H-K-P-NS1, HN-K-P-NS1,F-K-P-NS2, G-K-P-NS2, H-K-P-NS2, HN-K-P-NS2, F-K-P-L, G-K-P-L, H-K-P-L,HN-K-P-L, F-K-NS1-N, G-K-NS1-N, H-K-NS1-N, HN-K-NS1-N, F-K-NS1-M,G-K-NS1-M, H-K-NS1-M, HN-K-NS1-M, F-K-NS1-M2, G-K-NS1-M2, H-K-NS1-M2,HN-K-NS1-M2, F-K-NS1-P, G-K-NS1-P, H-K-NS1-P, HN-K-NS1-P, F-K-NS1-NS1,G-K-NS1-NS1, H-K-NS1-NS1, HN-K-NS1-NS1, F-K-NS1-NS2, G-K-NS1-NS2,H-K-NS1-NS2, HN-K-NS1-NS2, F-K-NS1-L, G-K-NS1-L, H-K-NS1-L, HN-K-NS1-L,F-K-NS2-N, G-K-NS2-N, H-K-NS2-N, HN-K-NS2-N, F-K-NS2-M, G-K-NS2-M,H-K-NS2-M, HN-K-NS2-M, F-K-NS2-M2, G-K-NS2-M2, H-K-NS2-M2, HN-K-NS2-M2,F-K-NS2-P, G-K-NS2-P, H-K-NS2-P, HN-K-NS2-P, F-K-NS2-NS1, G-K-NS2-NS1,H-K-NS2-NS1, HN-K-NS2-NS1, F-K-NS2-NS2, G-K-NS2-NS2, H-K-NS2-NS2,HN-K-NS2-NS2, F-K-NS2-L, G-K-NS2-L, H-K-NS2-L, HN-K-NS2-L, F-K-L-N,G-K-L-N, H-K-L-N, HN-K-L-N, F-K-L-M, G-K-L-M, H-K-L-M, HN-K-L-M,F-K-L-M2, G-K-L-M2, H-K-L-M2, HN-K-L-M2, F-K-L-P, G-K-L-P, H-K-L-P,HN-K-L-P, F-K-L-NS1, G-K-L-NS1, H-K-L-NS1, HN-K-L-NS1, F-K-L-NS2,G-K-L-NS2, H-K-L-NS2, HN-K-L-NS2, F-K-L-L, G-K-L-L, H-K-L-L, HN-K-L-L,F-N-K-N, G-N-K-N, H-N-K-N, HN-N-K-N, F-N-K-M, G-N-K-M, H-N-K-M,HN-N-K-M, F-N-K-M2, G-N-K-M2, H-N-K-M2, HN-N-K-M2, F-N-K-P, G-N-K-P,H-N-K-P, HN-N-K-P, F-N-K-NS1, G-N-K-NS1, H-N-K-NS1, HN-N-K-NS1,F-N-K-NS2, G-N-K-NS2, H-N-K-NS2, HN-N-K-NS2, F-N-K-L, G-N-K-L, H-N-K-L,HN-N-K-L, F-M-K-N, G-M-K-N, H-M-K-N, HN-M-K-N, F-M-K-M, G-M-K-M,H-M-K-M, HN-M-K-M, F-M-K-M2, G-M-K-M2, H-M-K-M2, HN-M-K-M2, F-M-K-P,G-M-K-P, H-M-K-P HN-M-K-P, F-M-K-NS1, G-M-K-NS1, H-M-K-NS1, HN-M-K-NS1,F-M-K-NS2, G-M-K-NS2, H-M-K-NS2, HN-M-K-NS2, F-M-K-L, G-M-K-L, H-M-K-L,HN-M-K-L, F-M2-K-N, G-M2-K-N, H-M2-K-N, HN-M2-K-N, F-M2-K-M, G-M2-K-M,H-M2-K-M, HN-M2-K-M, F-M2-K-M2, G-M2-K-M2, H-M2-K-M2, HN-M2-K-M2,F-M2-K-P, G-M2-K-P, H-M2-K-P HN-M2-K-P, F-M2-K-NS1, G-M2-K-NS1,H-M2-K-NS1, HN-M2-K-NS1, F-M2-K-NS2, G-M2-K-NS2, H-M2-K-NS2,HN-M2-K-NS2, F-M2-K-L, G-M2-K-L, H-M2-K-L, HN-M2-K-L, F-P-K-N, G-P-K-N,H-P-K-N, HN-P-K-N, F-P-K-M, G-P-K-M, H-P-K-M, HN-P-K-M, F-P-K-M2,G-P-K-M2, H-P-K-M2; HN-P-K-M2, F-P-K-P, G-P-K-P, H-P-K-P, HN-P-K-P,F-P-K-NS1, G-P-K-NS1, H-P-K-NS1, HN-P-K-NS1, F-P-K-NS2, G-P-K-NS2,H-P-K-NS2, HN-P-K-NS2, F-P-K-L, G-P-K-L, H-P-K-L, HN-P-K-L, F-NS1-K-N,G-NS1-K-N, H-NS1-K-N, HN-NS1-K-N, F-NS1-K-M, G-NS1-K-M, H-NS1-K-M;HN-NS1-K-M, F-NS1-K-M2, G-NS1-K-M2, H-NS1-K-M2; HN-NS1-K-M2, F-NS1-K-P,G-NS1-K-P, H-NS1-K-P, HN-NS1-K-P, F-NS1-K-NS1, G-NS1-K-NS1, H-NS1-K-NS1,HN-NS1-K-NS1, F-NS1-K-NS2, G-NS1-K-NS2, H-NS1-K-NS2, HN-NS1-K-NS2,F-NS1-K-L, G-NS1-K-L, H-NS1-K-L, HN-NS1-K-L, F-NS2-K-N, G-NS2-K-N,H-NS2-K-N, HN-NS2-K-N, F-NS2-K-M, G-NS2-K-M, H-NS2-K-M, HN-NS2-K-M,F-NS2-K-M2, G-NS2-K-M2, H-NS2-K-M2, HN-NS2-K-M2, F-NS2-K-P, G-NS2-K-P,H-NS2-K-P, HN-NS2-K-P, F-NS2-K-NS1, G-NS2-K-NS1, H-NS2-K-NS1,HN-NS2-K-NS1, F-NS2-K-NS2, G-NS2-K-NS2, H-NS2-K-NS2, HN-NS2-K-NS2,F-NS2-K-L, G-NS2-K-L, H-NS2-K-L, HN-NS2-K-L, F-C-M-M, G-C-M-M, H-C-M-M,HN-C-M-M, F-C-M-M2, G-C-M-M2, H-C-M-M2, HN-C-M-M2, F-C-M-N, G-C-M-N,H-C-M-N, HN-C-M-N, F-C-M-P, G-C-M-P, H-C-M-P, HN-C-M-P, F-C-M-NS1,G-C-M-NS1, H-C-M-NS1, HN-C-M-NS1, F-C-M-NS2, G-C-M-NS2, H-C-M-NS2,HN-C-M-NS2, F-C-M-L, G-C-M-L, H-C-M-L, HN-C-M-L, F-C-M2-M, G-C-M2-M,H-C-M2-M, HN-C-M2-M, F-C-M2-N, G-C-M2-N, H-C-M2-N, HN-C-M2-N, F-C-M2-P,G-C-M2-P, H-C-M2-P, HN-C-M2-P, F-C-M2-NS1, G-C-M2-NS1, H-C-M2-NS1,HN-C-M2-NS1, F-C-M2-NS2, G-C-M2-NS2, H-C-M2-NS2, HN-C-M2-NS2, F-C-M2-L,G-C-M2-L, H-C-M2-L, HN-C-M2-L, F-C-P-N, G-C-P-N, H-C-P-N, HN-C-P-N,F-C-P-M, G-C-P-M, H-C-P-M, HN-C-P-M, F-C-P-M2, G-C-P-M2, H-C-P-M2,HN-C-P-M2, F-C-P-P, G-C-P-P, H-C-P-P, HN-C-P-P, F-C-P-NS1, G-C-P-NS1,H-C-P-NS1, HN-C-P-NS1, F-C-P-NS2, G-C-P-NS2, H-C-P-NS2, HN-C-P-NS2,F-C-P-L, G-C-P-L, H-C-P-L, HN-C-P-L, F-C-NS1-N, G-C-NS1-N, H-C-NS1-N,HN-C-NS1-N, F-C-NS1-M, G-C-NS1-M, H-C-NS1-M, HN-C-NS1-M, F-C-NS1-M2,G-C-NS1-M2, H-C-NS1-M2, HN-C-NS1-M2, F-C-NS1-P, G-C-NS1-P, H-C-NS1-P,HN-C-NS1-P, F-C-NS1-NS1, G-C-NS1-NS1, H-C-NS1-NS1, HN-C-NS1-NS1,F-C-NS1-NS2, G-C-NS1-NS2, H-C-NS1-NS2, HN-C-NS1-NS2, F-C-NS1-L,G-C-NS1-L, H-C-NS1-L, HN-C-NS1-L, F-C-NS2-N, G-C-NS2-N, H-C-NS2-N,HN-C-NS2-N, F-C-NS2-M, G-C-NS2-M, H-C-NS2-M, HN-C-NS2-M, F-C-NS2-M2,G-C-NS2-M2, H-C-NS2-M2, HN-C-NS2-M2, F-C-NS2-P, G-C-NS2-P, H-C-NS2-P,HN-C-NS2-P, F-C-NS2-NS1, G-C-NS2-NS1, H-C-NS2-NS1, HN-C-NS2-NS1,F-C-NS2-NS2, G-C-NS2-NS2, H-C-NS2-NS2, HN-C-NS2-NS2, F-C-NS2-L,G-C-NS2-L, H-C-NS2-L, HN-C-NS2-L, F-C-L-N, G-C-L-N, H-C-L-N, HN-C-L-N,F-C-L-M, G-C-L-M, H-C-L-M, HN-C-L-M, F-C-L-M2, G-C-L-M2, H-C-L-M2,HN-C-L-M2, F-C-L-P, G-C-L-P, H-C-L-P, HN-C-L-P, F-C-L-NS1, G-C-L-NS1,H-C-L-NS1, HN-C-L-NS1, F-C-L-NS2, G-C-L-NS2, H-C-L-NS2, HN-C-L-NS2,F-C-L-L, G-C-L-L, H-C-L-L, HN-C-L-L, F-N-C-N, G-N-C-N, H-N-C-N,HN-N-C-N, F-N-C-M, G-N-C-M, H-N-C-M, HN-N-C-M, F-N-C-M2, G-N-C-M2,H-N-C-M2, HN-N-C-M2, F-N-C-P, G-N-C-P, H-N-C-P, HN-N-C-P, F-N-C-NS1,G-N-C-NS1, H-N-C-NS1, HN-N-C-NS1, F-N-C-NS2, G-N-C-NS2, H-N-C-NS2,HN-N-C-NS2, F-N-C-L, G-N-C-L, H-N-C-L, HN-N-C-L, F-M-C-N, G-M-C-N,H-M-C-N, HN-M-C-N, F-M-C-M, G-M-C-M, H-M-C-M, HN-M-C-M, F-M-C-M2,G-M-C-M2, H-M-C-M2, HN-M-C-M2, F-M-C-P, G-M-C-P, H-M-C-P HN-M-C-P,F-M-C-NS1, G-M-C-NS1, H-M-C-NS1, HN-M-C-NS1, F-M-C-NS2, G-M-C-NS2,H-M-C-NS2, HN-M-C-NS2, F-M-C-L, G-M-C-L, H-M-C-L, HN-M-C-L, F-M2-C-N,G-M2-C-N, H-M2-C-N, HN-M2-C-N, F-M2-C-M, G-M2-C-M, H-M2-C-M, HN-M2-C-M,F-M2-C-M2, G-M2-C-M2, H-M2-C-M2, HN-M2-C-M2, F-M2-C-P, G-M2-C-P,H-M2-C-P HN-M2-C-P, F-M2-C-NS1, G-M2-C-NS1, H-M2-C-NS1, HN-M2-C-NS1,F-M2-C-NS2, G-M2-C-NS2, H-M2-C-NS2, HN-M2-C-NS2, F-M2-C-L, G-M2-C-L,H-M2-C-L, HN-M2-C-L, F-P-C-N, G-P-C-N, H-P-C-N, HN-P-C-N, F-P-C-M,G-P-C-M, H-P-C-M, HN-P-C-M, F-P-C-M2, G-P-C-M2, H-P-C-M2; HN-P-C-M2,F-P-C-P, G-P-C-P, H-P-C-P, HN-P-C-P, F-P-C-NS1, G-P-C-NS1, H-P-C-NS1,HN-P-C-NS1, F-P-C-NS2, G-P-C-NS2, H-P-C-NS2, HN-P-C-NS2, F-P-C-L,G-P-C-L, H-P-C-L, HN-P-C-L, F-NS1-C-N, G-NS1-C-N, H-NS1-C-N, HN-NS1-C-N,F-NS1-C-M, G-NS1-C-M, H-NS1-C-M; HN-NS1-C-M, F-NS1-C-M2, G-NS1-C-M2,H-NS1-C-M2; HN-NS1-C-M2, F-NS1-C-P, G-NS1-C-P, H-NS1-C-P, HN-NS1-C-P,F-NS1-C-NS1, G-NS1-C-NS1, H-NS1-C-NS1, HN-NS1-C-NS1, F-NS1-C-NS2,G-NS1-C-NS2, H-NS1-C-NS2, HN-NS1-C-NS2, F-NS1-C-L, G-NS1-C-L, H-NS1-C-L,HN-NS1-C-L, F-NS2-C-N, G-NS2-C-N, H-NS2-C-N, HN-NS2-C-N, F-NS2-C-M,G-NS2-C-M, H-NS2-C-M, HN-NS2-C-M, F-NS2-C-M2, G-NS2-C-M2, H-NS2-C-M2,HN-NS2-C-M2, F-NS2-C-P, G-NS2-C-P, H-NS2-C-P, HN-NS2-C-P, F-NS2-C-NS1,G-NS2-C-NS1, H-NS2-C-NS1, HN-NS2-C-NS1, F-NS2-C-NS2, G-NS2-C-NS2,H-NS2-C-NS2, HN-NS2-C-NS2, F-NS2-C-L, G-NS2-C-L, H-NS2-C-L, HN-NS2-C-L,F-L-K-N, G-L-K-N, H-L-K-N, HN-L-K-N, F-L-K-M, G-L-K-M, H-L-K-M,HN-L-K-M, F-L-K-M2, G-L-K-M2, H-L-K-M2, HN-L-K-M2, F-L-K-P, G-L-K-P,H-L-K-P, HN-L-K-P, F-L-K-NS1, G-L-K-NS1, H-L-K-NS1, HN-L-K-NS1,F-L-K-NS2, G-L-K-NS2, H-L-K-NS2, HN-L-K-NS2, F-L-K-L, G-L-K-L, H-L-K-L,HN-L-K-L, F-L-C-N, G-L-C-N, H-L-C-N, HN-L-C-N, F-L-C-M, G-L-C-M,H-L-C-M, HN-L-C-M, F-L-C-M2, G-L-C-M2, H-L-C-M2, HN-L-C-M2, F-L-C-P,G-L-C-P, H-L-C-P, HN-L-C-P, F-L-C-NS1, G-L-C-NS1, H-L-C-NS1, HN-L-C-NS1,F-L-C-NS2, G-L-C-NS2, H-L-C-NS2, HN-L-C-NS2, F-L-C-L, G-L-C-L, H-L-C-L,HN-L-C-L, F-K-N-K-N, G-K-N-K-N, H-K-N-K-N, HN-K-N-K-N, F-K-M-K-N,G-K-M-K-N, H-K-M-K-N, HN-K-M-K-N, F-K-M2-K-N, G-K-M2-K-N, H-K-M2-K-N,HN-K-M2-K-N, F-K-P-K-N, G-K-P-K-N, H-K-P-K-N, HN-K-P-K-N, F-K-NS1-K-N,G-K-NS1-K-N, H-K-NS1-K-N, HN-K-NS1-K-N, F-K-NS2-K-N, G-K-NS2-K-N,H-K-NS2-K-N, HN-K-NS2-K-N, F-K-L-K-N, G-K-L-K-N, H-K-L-K-N, HN-K-L-K-N,F-K-N-K-M, G-K-N-K-M, H-K-N-K-M, HN-K-N-K-M, F-K-M-K-M, G-K-M-K-M,H-K-M-K-M, HN-K-M-K-M, F-K-M2-K-M, G-K-M2-K-M, H-K-M2-K-M, HN-K-M2-K-M,F-K-P-K-M, G-K-P-K-M, H-K-P-K-M, HN-K-P-K-M, F-K-NS1-K-M, G-K-NS1-K-M,H-K-NS1-K-M, HN-K-NS1-K-M, F-K-NS2-K-M, G-K-NS2-K-M, H-K-NS2-K-M,HN-K-NS2-K-M, F-K-L-K-M, G-K-L-K-M, H-K-L-K-M, HN-K-L-K-M, F-K-N-K-M2,G-K-N-K-M2, H-K-N-K-M2, HN-K-N-K-M2, F-K-M-K-M2, G-K-M-K-M2, H-K-M-K-M2,HN-K-M-K-M2, F-K-M2-K-M2, G-K-M2-K-M2, H-K-M2-K-M2, HN-K-M2-K-M2,F-K-P-K-M2, G-K-P-K-M2, H-K-P-K-M2, HN-K-P-K-M2, F-K-NS1-K-M2,G-K-NS1-K-M2, H-K-NS1-K-M2, HN-K-NS1-K-M2, F-K-NS2-K-M2, G-K-NS2-K-M2,H-K-NS2-K-M2, HN-K-NS2-K-M2, F-K-L-K-M2, G-K-L-K-M2, H-K-L-K-M2,HN-K-L-K-M2, F-K-N-K-P, G-K-N-K-P, H-K-N-K-P, HN-K-N-K-P, F-K-M-K-P,G-K-M-K-P, H-K-M-K-P, HN-K-M-K-P, F-K-M2-K-P, G-K-M2-K-P, H-K-M2-K-P,HN-K-M2-K-P, F-K-P-K-P, G-K-P-K-P, H-K-P-K-P, HN-K-P-K-P, F-K-NS1-K-P,G-K-NS1-K-P, H-K-NS1-K-P, HN-K-NS1-K-P, F-K-NS2-K-P, G-K-NS2-K-P,H-K-NS2-K-P, HN-K-NS2-K-P, F-K-L-K-P, G-K-L-K-P, H-K-L-K-P, HN-K-L-K-P,F-K-N-K-NS1, G-K-N-K-NS1, H-K-N-K-NS1, HN-K-N-K-NS1, F-K-M-K-NS1,G-K-M-K-NS1, H-K-M-K-NS1, HN-K-M-K-NS1, F-K-M2-K-NS1, G-K-M2-K-NS1,H-K-M2-K-NS1, HN-K-M2-K-NS1, F-K-P-K-NS1, G-K-P-K-NS1, H-K-P-K-NS1,HN-K-P-K-NS1, F-K-NS1-K-NS1, G-K-NS1-K-NS1, H-K-NS1-K-NS1,HN-K-NS1-K-NS1, F-K-NS2-K-NS1, G-K-NS2-K-NS1, H-K-NS2-K-NS1,HN-K-NS2-K-NS1, F-K-L-K-NS1, G-K-L-K-NS1, H-K-L-K-NS1, HN-K-L-K-NS1,F-K-N-K-NS2, G-K-N-K-NS2, H-K-N-K-NS2, HN-K-N-K-NS2, F-K-M-K-NS2,G-K-M-K-NS2, H-K-M-K-NS2, HN-K-M-K-NS2, F-K-M2-K-NS2, G-K-M2-K-NS2,H-K-M2-K-NS2, HN-K-M2-K-NS2, F-K-P-K-NS2, G-K-P-K-NS2, H-K-P-K-NS2,HN-K-P-K-NS2, F-K-NS1-K-NS2 G-K-NS1-K-NS2, H-K-NS1-K-NS2,HN-K-NS1-K-NS2, F-K-NS2-K-NS2 G-K-NS2-K-NS2, H-K-NS2-K-NS2,HN-K-NS2-K-NS2, F-K-L-K-NS2, G-K-L-K-NS2, H-K-L-K-NS2, HN-K-L-K-NS2,F-K-N-K-L, G-K-N-K-L, H-K-N-K-L, HN-K-N-K-K, F-K-M-K-L, G-K-M-K-L,H-K-M-K-L, HN-K-M-K-L, F-K-M2-K-L, G-K-M2-K-L, H-K-M2-K-L, HN-K-M2-K-L,F-K-P-K-L, G-K-P-K-L, H-K-P-K-L, HN-K-P-K-L, F-K-NS1-K-L, G-K-NS1-K-L,H-K-NS1-K-L, HN-K-NS1-K-L, F-K-NS2-K-L, G-K-NS2-K-L, H-K-NS2-K-L,HN-K-NS2-K-L, F-K-L-K-L, G-K-L-K-L, H-K-L-K-L, HN-K-L-K-L, N-K-N-F,N-K-N-G, N-K-N-H, N-K-N-HN, M-K-N-F, M-K-N-G, M-K-N-H, M-K-N-HN,M2-K-N-F, M2-K-N-G, M2-K-N-H, M2-K-N-HN, P-K-N-F, P-K-N-G, P-K-N-H,P-K-N-HN, NS1-K-N-F, NS1-K-N-G, NS1-K-N-H, NS1-K-N-HN, NS2-K-N-F,NS2-K-N-G, NS2-K-N-H, NS2-K-N-HN, L-K-N-F, L-K-N-G, L-K-N-H, L-K-N-HN,N-K-M-F, N-K-M-G, N-K-M-H, N-K-M-HN, M-K-M-F, M-K-M-G, M-K-M-H,M-K-M-HN, M2-K-M-F, M2-K-M-G, M2-K-M-H, M2-K-M-HN, P-K-M-F, P-K-M-G,P-K-M-H, P-K-M-HN, NS1-K-M-F, NS1-K-M-G, NS1-K-M-H, NS1-K-M-HN,NS2-K-M-F, NS2-K-M-G, NS2-K-M-H, NS2-K-M-HN, L-K-M-F, L-K-M-G, L-K-M-H,L-K-M-HN, N-K-M2-F, N-K-M2-G, N-K-M2-H, N-K-M2-HN, M-K-M2-F, M-K-M2-G,M-K-M2-H, M-K-M2-HN, M2-K-M2-F, M2-K-M2-G, M2-K-M2-H, M2-K-M2-HN,P-K-M2-F, P-K-M2-G, P-K-M2-H, P-K-M2-HN, NS1-K-M2-F, NS1-K-M2-G,NS1-K-M2-H, NS1-K-M2-HN, NS2-K-M2-F, NS2-K-M2-G, NS2-K-M2-H,NS2-K-M2-HN, L-K-M2-F, L-K-M2-G, L-K-M2-H, L-K-M2-HN, N-K-P-F, N-K-P-G,N-K-P-H, N-K-P-HN, M-K-P-F, M-K-P-G, M-K-P-H, M-K-P-HN, M2-K-P-F,M2-K-P-G, M2-K-P-H, M2-K-P-HN, P-K-P-F, P-K-P-G, P-K-P-H, P-K-P-HN,NS1-K-P-F, NS1-K-P-G, NS1-K-P-H, NS1-K-P-HN, NS2-K-P-F, NS2-K-P-G,NS2-K-P-H, NS2-K-P-HN, L-K-P-F, L-K-P-G, L-K-P-H, L-K-P-HN, N-K-NS1-F,N-K-NS1-G, N-K-NS1-H, N-K-NS1-HN, M-K-NS1-F, M-K-NS1-G, M-K-NS1-H,M-K-NS1-HN, M2-K-NS1-F, M2-K-NS1-G, M2-K-NS1-H, M2-K-NS1-HN, P-K-NS1-F,P-K-NS1-G, P-K-NS1-H, P-K-NS1-HN, NS1-K-NS1-F, NS1-K-NS1-G, NS1-K-NS1-H,NS1-K-NS1-HN, NS2-K-NS1-F, NS2-K-NS1-G, NS2-K-NS1-H, NS2-K-NS1-HN,L-K-NS1-F, L-K-NS1-G, L-K-NS1-H, L-K-NS1-HN, N-K-NS2-F, N-K-NS2-G,N-K-NS2-H, N-K-NS2-HN, M-K-NS2-F, M-K-NS2-G, M-K-NS2-H, M-K-NS2-HN,M2-K-NS2-F, M2-K-NS2-G, M2-K-NS2-H, M2-K-NS2-HN, P-K-NS2-F, P-K-NS2-G,P-K-NS2-H, P-K-NS2-HN, NS1-K-NS2-F, NS1-K-NS2-G, NS1-K-NS2-H,NS1-K-NS2-HN, NS2-K-NS2-F, NS2-K-NS2-G, NS2-K-NS2-H, NS2-K-NS2-HN,L-K-NS2-F, L-K-NS2-G, L-K-NS2-H, L-K-NS2-HN, N-K-L-F, N-K-L-G, N-K-L-H,N-K-L-HN, M-K-L-F, M-K-L-G, M-K-L2-H, M-K-L-HN, M2-K-L-F, M2-K-L-G,M2-K-L-H, M2-K-L-HN, P-K-L-F, P-K-L-G, P-K-L-H, P-K-L-HN, NS1-K-L-F,NS1-K-L-G, NS1-K-L-H, NS1-K-L-HN, NS2-K-L-F, NS2-K-L-G, NS2-K-L-H,NS2-K-L-HN, L-K-L-F, L-K-L-G, L-K-L-H, L-K-L-HN, N-N-K-F, N-N-K-G,N-N-K-H, N-N-K-HN, N-M-K-F, N-M-K-G, N-M-K-H, N-M-K-HN, N-M2-K-F,N-M2-K-G, N-M2-K-H, N-M2-K-HN, N-P-K-F, N-P-K-G, N-P-K-H, N-P-K-HN,N-NS1-K-F, N-NS1-K-G, N-NS1-K-H, N-NS1-K-HN, N-NS2-K-F, N-NS2-K-G,N-NS2-K-H, N-NS2-K-HN, N-L-K-F, N-L-K-G, N-L-K-H, N-L-K-HN, M-N-K-F,M-N-K-G, M-N-K-H, M-N-K-HN, M-M-K-F, M-M-K-G, M-M-K-H, M-M-K-HN,M-M2-K-F, M-M2-K-G, M-M2-K-H, M-M2-K-HN, M-P-K-F, M-P-K-G, M-P-K-H,M-P-K-HN, M-NS1-K-F, M-NS1-K-G, M-NS1-K-H, M-NS1-K-HN, M-NS2-K-F,M-NS2-K-G, M-NS2-K-H, M-NS2-K-HN, M-L-K-F, M-L-K-G, M-L-K-H, M-L-K-HN,M2-N-K-F, M2-N-K-G, M2-N-K-H, M2-N-K-HN, M2-M-K-F, M2-M-K-G, M2-M-K-H,M2-M-K-HN, M2-M2-K-F, M2-M2-K-G, M2-M2-K-H, M2-M2-K-HN, M2-P-K-F,M2-P-K-G, M2-P-K-H, M2-P-K-HN, M2-NS1-K-F, M2-NS1-K-G, M2-NS1-K-H,M2-NS1-K-HN, M2-NS2-K-F, M2-NS2-K-G, M2-NS2-K-H, M2-NS2-K-HN, M2-L-K-F,M2-L-K-G, M2-L-K-H, M2-L-K-HN, P-N-K-F, P-N-K-G, P-N-K-H, P-N-K-HN,P-M-K-F, P-M-K-G, P-M-K-H, P-M-K-HN, P-M2-K-F, P-M2-K-G, P-M2-K-H,P-M2-K-HN, P-P-K-F, P-P-K-G, P-P-K-H, P-P-K-HN, P-NS1-K-F, P-NS1-K-G,P-NS1-K-H, P-NS1-K-HN, P-NS2-K-F, P-NS2-K-G, P-NS2-K-H, P-NS2-K-HN,P-L-K-F, P-L-K-G, P-L-K-H, P-L-K-HN, NS1-N-K-F, NS1-N-K-G, NS1-N-K-H,NS1-N-K-HN, NS1-M-K-F, NS1-M-K-G, NS1-M-K-H, NS1-M-K-HN, NS1-M2-K-F,NS1-M2-K-G, NS1-M2-K-H, NS1-M2-K-HN, NS1-P-K-F, NS1-P-K-G, NS1-P-K-H,NS1-P-K-HN, NS1-NS1-K-F, NS1-NS1-K-G, NS1-NS1-K-H, NS1-NS1-K-HN,NS1-NS2-K-F, NS1-NS2-K-G, NS1-NS2-K-H, NS1-NS2-K-HN, NS1-L-K-F,NS1-L-K-G, NS1-L-K-H, NS1-L-K-HN, NS2-N-K-F, NS2-N-K-G, NS2-N-K-H,NS2-N-K-HN, NS2-M-K-F, NS2-M-K-G, NS2-M-K-H, NS2-M-K-HN, NS2-M2-K-F,NS2-M2-K-G, NS2-M2-K-H, NS2-M2-K-HN, NS2-P-K-F, NS2-P-K-G, NS2-P-K-H,NS2-P-K-HN, NS2-NS1-K-F, NS2-NS1-K-G, NS2-NS1-K-H, NS2-NS1-K-HN,NS2-NS2-K-F, NS2-NS2-K-G, NS2-NS2-K-H, NS2-NS2-K-HN, NS2-L-K-F,NS2-L-K-G, NS2-L-K-H, NS2-L-K-HN, L-N-K-F, L-N-K-G, L-N-K-H, L-N-K-HN,L-M-K-F, L-M-K-G, L-M-K-H, L-M-K-HN, L-M2-K-F, L-M2-K-G, L-M2-K-H,L-M2-K-HN, L-L-K-F, L-P-K-G, L-P-K-H, L-P-K-HN, L-NS1-K-F, L-NS1-K-G,L-NS1-K-H, L-NS1-K-HN, L-NS2-K-F, L-NS2-K-G, L-NS2-K-H, L-NS2-K-HN,L-L-K-F, L-L-K-G, L-L-K-H, L-L-K-HN, N-K-N-K-F, N-K-N-K-G, N-K-N-K-H,N-K-N-K-HN, N-K-M-K-F, N-K-M-K-G, N-K-M-K-H, N-K-M-K-HN, N-K-M2-K-F,N-K-M2-K-G, N-K-M2-K-H, N-K-M2-K-HN, N-K-P-K-F, N-K-P-K-G, N-K-P-K-H,N-K-P-K-HN, N-K-NS1-K-F, N-K-NS1-K-G, N-K-NS1-K-H, N-K-NS1-K-HN,N-K-NS2-K-F, N-K-NS2-K-G, N-K-NS2-K-H, N-K-NS2-K-HN, N-K-L-K-F,N-K-L-K-G, N-K-L-K-H, N-K-L-K-HN, M-K-N-K-F, M-K-N-K-G, M-K-N-K-H,M-K-N-K-HN, M-K-M-K-F, M-K-M-K-G, M-K-M-K-H, M-K-M-K-HN, M-K-M2-K-F,M-K-M2-K-G, M-K-M2-K-H, M-K-M2-K-HN, M-K-P-K-F, M-K-P-K-G, M-K-P-K-H,M-K-P-K-HN, M-K-NS1-K-F, M-K-NS1-K-G, M-K-NS1-K-H, M-K-NS1-K-HN,M-K-NS2-K-F, M-K-NS2-K-G, M-K-NS2-K-H, M-K-NS2-K-HN, M-K-L-K-F,M-K-L-K-G, M-K-L-K-H, M-K-L-K-HN, M2-K-N-K-F, M2-K-N-K-G, M2-K-N-K-H,M2-K-N-K-HN, M2-K-M-K-F, M2-K-M-K-G, M2-K-M-K-H, M2-K-M-K-HN,M2-K-M2-K-F, M2-K-M2-K-G, M2-K-M2-K-H, M2-K-M2-K-HN, M2-K-P-K-F,M2-K-P-K-G, M2-K-P-K-H, M2-K-P-K-HN, M2-K-NS1-K-F, M2-K-NS1-K-G,M2-K-NS1-K-H, M2-K-NS1-K-HN, M2-K-NS2-K-F, M2-K-NS2-K-G, M2-K-NS2-K-H,M2-K-NS2-K-HN, M2-K-L-K-F, M2-K-L-K-G, M2-K-L-K-H, M2-K-L-K-HN,P-K-N-K-F, P-K-N-K-G, P-K-N-K-H, P-K-N-K-HN, P-K-M-K-F, P-K-M-K-G,P-K-M-K-H, P-K-M-K-HN, P-K-M2-K-F, P-K-M2-K-G, P-K-M2-K-H, P-K-M2-K-HN,P-K-P-K-F, P-K-P-K-G, P-K-P-K-H, P-K-P-K-HN, P-K-NS1-K-F, P-K-NS1-K-G,P-K-NS1-K-H, P-K-NS1-K-HN, P-K-NS2-K-F, P-K-NS2-K-G, P-K-NS2-K-H,P-K-NS2-K-HN, P-K-L-K-F, P-K-L-K-G, P-K-L-K-H, P-K-L-K-HN, NS1-K-N-K-F,NS1-K-N-K-G, NS1-K-N-K-H, NS1-K-N-K-HN, NS1-K-M-K-F, NS1-K-M-K-G,NS1-K-M-K-H, NS1-K-M-K-HN, NS1-K-M2-K-F, NS1-K-M2-K-G, NS1-K-M2-K-H,NS1-K-M2-K-HN, NS1-K-P-K-F, NS1-K-P-K-G, NS1-K-P-K-H, NS1-K-P-K-HN,NS1-K-NS1-K-F, NS1-K-NS1-K-G, NS1-K-NS1-K-H, NS1-K-NS1-K-HN,NS1-K-NS2-K-F, NS1-K-NS2-K-G, NS1-K-NS2-K-H, NS1-K-NS2-K-HN,NS1-K-L-K-F, NS1-K-L-K-G, NS1-K-L-K-H, NS1-K-L-K-HN, NS2-K-N-K-F,NS2-K-N-K-G, NS2-K-N-K-H, NS2-K-N-K-HN, NS2-K-M-K-F, NS2-K-M-K-G,NS2-K-M-K-H, NS2-K-M-K-HN, NS2-K-M2-K-F, NS2-K-M2-K-G, NS2-K-M2-K-H,NS2-K-M2-K-HN, NS2-K-P-K-F, NS2-K-P-K-G, NS2-K-P-K-H, NS2-K-P-K-HN,NS2-K-NS1-K-F, NS2-K-NS1-K-G, NS2-K-NS1-K-H, NS2-K-NS1-K-HN,NS2-K-NS2-K-F, NS2-K-NS2-K-G, NS2-K-NS2-K-H, NS2-K-NS2-K-HN,NS2-K-L-K-F, NS2-K-L-K-G, NS2-K-L-K-H, NS2-K-L-K-HN, L-K-N-K-F,L-K-N-K-G, L-K-N-K-H, L-K-N-K-HN, L-K-M-K-F, L-K-M-K-G, L-K-M-K-H,L-K-M-K-HN, L-K-M2-K-F, L-K-M2-K-G, L-K-M2-K-H, L-K-M2-K-HN, L-K-P-K-F,L-K-P-K-G, L-K-P-K-H, L-K-P-K-HN, L-K-NS1-K-F, L-K-NS1-K-G, L-K-NS1-K-H,L-K-NS1-K-HN, L-K-NS2-K-F, L-K-NS2-K-G, L-K-NS2-K-H, L-K-NS2-K-HN,L-K-L-K-F, L-K-L-K-G, L-K-L-K-H, or L-K-L-K-HN, F-C-N-K-N, G-C-N-K-N,H-C-N-K-N, HN-C-N-K-N, F-C-M-K-N, G-C-M-K-N, H-C-M-K-N, HN-C-M-K-N,F-C-M2-K-N, G-C-M2-K-N, H-C-M2-K-N, HN-C-M2-K-N, F-C-P-K-N, G-C-P-K-N,H-C-P-K-N, HN-C-P-K-N, F-C-NS1-K-N, G-C-NS1-K-N, H-C-NS1-K-N,HN-C-NS1-K-N, F-C-NS2-K-N, G-C-NS2-K-N, H-C-NS2-K-N, HN-C-NS2-K-N,F-C-L-K-N, G-C-L-K-N, H-C-L-K-N, HN-C-L-K-N, F-C-N-K-M, G-C-N-K-M,H-C-N-K-M, HN-C-N-K-M, F-C-M-K-M, G-C-M-K-M, H-C-M-K-M, HN-C-M-K-M,F-C-M2-K-M, G-C-M2-K-M, H-C-M2-K-M, HN-C-M2-K-M, F-C-P-K-M, G-C-P-K-M,H-C-P-K-M, HN-C-P-K-M, F-C-NS1-K-M, G-C-NS1-K-M, H-C-NS1-K-M,HN-C-NS1-K-M, F-C-NS2-K-M, G-C-NS2-K-M, H-C-NS2-K-M, HN-C-NS2-K-M,F-C-L-K-M, G-C-L-K-M, H-C-L-K-M, HN-C-L-K-M, F-C-N-K-M2, G-C-N-K-M2,H-C-N-K-M2, HN-C-N-K-M2, F-C-M-K-M2, G-C-M-K-M2, H-C-M-K-M2,HN-C-M-K-M2, F-C-M2-K-M2, G-C-M2-K-M2, H-C-M2-K-M2, HN-C-M2-K-M2,F-C-P-K-M2, G-C-P-K-M2, H-C-P-K-M2, HN-C-P-K-M2, F-C-NS1-K-M2,G-C-NS1-K-M2, H-C-NS1-K-M2, HN-C-NS1-K-M2, F-C-NS2-K-M2, G-C-NS2-K-M2,H-C-NS2-K-M2, HN-C-NS2-K-M2, F-C-L-K-M2, G-C-L-K-M2, H-C-L-K-M2,HN-C-L-K-M2, F-C-N-K-P, G-C-N-K-P, H-C-N-K-P, HN-C-N-K-P, F-C-M-K-P,G-C-M-K-P, H-C-M-K-P, HN-C-M-K-P, F-C-M2-K-P, G-C-M2-K-P, H-C-M2-K-P,HN-C-M2-K-P, F-C-P-K-P, G-C-P-K-P, H-C-P-K-P, HN-C-P-K-P, F-C-NS1-K-P,G-C-NS1-K-P, H-C-NS1-K-P, HN-C-NS1-K-P, F-C-NS2-K-P, G-C-NS2-K-P,H-C-NS2-K-P, HN-C-NS2-K-P, F-C-L-K-P, G-C-L-K-P, H-C-L-K-P, HN-C-L-K-P,F-C-N-K-NS1, G-C-N-K-NS1, H-C-N-K-NS1, HN-C-N-K-NS1, F-C-M-K-NS1,G-C-M-K-NS1, H-C-M-K-NS1, HN-C-M-K-NS1, F-C-M2-K-NS1, G-C-M2-K-NS1,H-C-M2-K-NS1, HN-C-M2-K-NS1, F-C-P-K-NS1, G-C-P-K-NS1, H-C-P-K-NS1,HN-C-P-K-NS1, F-C-NS1-K-NS1, G-C-NS1-K-NS1, H-C-NS1-K-NS1,HN-C-NS1-K-NS1, F-C-NS2-K-NS1, G-C-NS2-K-NS1, H-C-NS2-K-NS1,HN-C-NS2-K-NS1, F-C-L-K-NS1, G-C-L-K-NS1, H-C-L-K-NS1, HN-C-L-K-NS1,F-C-N-K-NS2, G-C-N-K-NS2, H-C-N-K-NS2, HN-C-N-K-NS2, F-C-M-K-NS2,G-C-M-K-NS2, H-C-M-K-NS2, HN-C-M-K-NS2, F-C-M2-K-NS2, G-C-M2-K-NS2,H-C-M2-K-NS2, HN-C-M2-K-NS2, F-C-P-K-NS2, G-C-P-K-NS2, H-C-P-K-NS2,HN-C-P-K-NS2, F-C-NS1-K-NS2 G-C-NS1-K-NS2, H-C-NS1-K-NS2,HN-C-NS1-K-NS2, F-C-NS2-K-NS2 G-C-NS2-K-NS2, H-C-NS2-K-NS2,HN-C-NS2-K-NS2, F-C-L-K-NS2, G-C-L-K-NS2, H-C-L-K-NS2, HN-C-L-K-NS2,F-C-N-K-L, G-C-N-K-L, H-C-N-K-L, HN-C-N-K-L, F-C-M-K-L, G-C-M-K-L,H-C-M-K-L, HN-C-M-K-L, F-C-M2-K-L, G-C-M2-K-L, H-C-M2-K-L, HN-C-M2-K-L,F-C-P-K-L, G-C-P-K-L, H-C-P-K-L, HN-C-P-K-L, F-C-NS1-K-L, G-C-NS1-K-L,H-C-NS1-K-L, HN-C-NS1-K-L, F-C-NS2-K-L, G-C-NS2-K-L, H-C-NS2-K-L,HN-C-NS2-K-L, F-C-L-K-L, G-C-L-K-L, H-C-L-K-L, HN-C-L-K-L, N-C-N-F,N-C-N-G, N-C-N-H, N-C-N-HN, M-C-N-F, M-C-N-G, M-C-N-H, M-C-N-HN,M2-C-N-F, M2-C-N-G, M2-C-N-H, M2-C-N-HN, P-C-N-F, P-C-N-G, P-C-N-H,P-C-N-HN, NS1-C-N-F, NS1-C-N-G, NS1-C-N-H, NS1-C-N-HN, NS2-C-N-F,NS2-C-N-G, NS2-C-N-H, NS2-C-N-HN, L-C-N-F, L-C-N-G, L-C-N-H, L-C-N-HN,N-C-M-F, N-C-M-G, N-C-M-H, N-C-M-HN, M-C-M-F, M-C-M-G, M-C-M-H,M-C-M-HN, M2-C-M-F, M2-C-M-G, M2-C-M-H, M2-C-M-HN, P-C-M-F, P-C-M-G,P-C-M-H, P-C-M-HN, NS1-C-M-F, NS1-C-M-G, NS1-C-M-H, NS1-C-M-HN,NS2-C-M-F, NS2-C-M-G, NS2-C-M-H, NS2-C-M-HN, L-C-M-F, L-C-M-G, L-C-M-H,L-C-M-HN, N-C-M2-F, N-C-M2-G, N-C-M2-H, N-C-M2-HN, M-C-M2-F, M-C-M2-G,M-C-M2-H, M-C-M2-HN, M2-C-M2-F, M2-C-M2-G, M2-C-M2-H, M2-C-M2-HN,P-C-M2-F, P-C-M2-G, P-C-M2-H, P-C-M2-HN, NS1-C-M2-F, NS1-C-M2-G,NS1-C-M2-H, NS1-C-M2-HN, NS2-C-M2-F, NS2-C-M2-G, NS2-C-M2-H,NS2-C-M2-HN, L-C-M2-F, L-C-M2-G, L-C-M2-H, L-C-M2-HN, N-C-P-F, N-C-P-G,N-C-P-H, N-C-P-HN, M-C-P-F, M-C-P-G, M-C-P-H, M-C-P-HN, M2-C-P-F,M2-C-P-G, M2-C-P-H, M2-C-P-HN, P-C-P-F, P-C-P-G, P-C-P-H, P-C-P-HN,NS1-C-P-F, NS1-C-P-G, NS1-C-P-H, NS1-C-P-HN, NS2-C-P-F, NS2-C-P-G,NS2-C-P-H, NS2-C-P-HN, L-C-P-F, L-C-P-G, L-C-P-H, L-C-P-HN, N-C-NS1-F,N-C-NS1-G, N-C-NS1-H, N-C-NS1-HN, M-C-NS1-F, M-C-NS1-G, M-C-NS1-H,M-C-NS1-HN, M2-C-NS1-F, M2-C-NS1-G, M2-C-NS1-H, M2-C-NS1-HN, P-C-NS1-F,P-C-NS1-G, P-C-NS1-H, P-C-NS1-HN, NS1-C-NS1-F, NS1-C-NS1-G, NS1-C-NS1-H,NS1-C-NS1-HN, NS2-C-NS1-F, NS2-C-NS1-G, NS2-C-NS1-H, NS2-C-NS1-HN,L-C-NS1-F, L-C-NS1-G, L-C-NS1-H, L-C-NS1-HN, N-C-NS2-F, N-C-NS2-G,N-C-NS2-H, N-C-NS2-HN, M-C-NS2-F, M-C-NS2-G, M-C-NS2-H, M-C-NS2-HN,M2-C-NS2-F, M2-C-NS2-G, M2-C-NS2-H, M2-C-NS2-HN, P-C-NS2-F, P-C-NS2-G,P-C-NS2-H, P-C-NS2-HN, NS1-C-NS2-F, NS1-C-NS2-G, NS1-C-NS2-H,NS1-C-NS2-HN, NS2-C-NS2-F, NS2-C-NS2-G, NS2-C-NS2-H, NS2-C-NS2-HN,L-C-NS2-F, L-C-NS2-G, L-C-NS2-H, L-C-NS2-HN, N-C-L-F, N-C-L-G, N-C-L-H,N-C-L-HN, M-C-L-F, M-C-L-G, M-C-L2-H, M-C-L-HN, M2-C-L-F, M2-C-L-G,M2-C-L-H, M2-C-L-HN, P-C-L-F, P-C-L-G, P-C-L-H, P-C-L-HN, NS1-C-L-F,NS1-C-L-G, NS1-C-L-H, NS1-C-L-HN, NS2-C-L-F, NS2-C-L-G, NS2-C-L-H,NS2-C-L-HN, L-C-L-F, L-C-L-G, L-C-L-H, L-C-L-HN, N-N-C-F, N-N-C-G,N-N-C-H, N-N-C-HN, N-M-C-F, N-M-C-G, N-M-C-H, N-M-C-HN, N-M2-C-F,N-M2-C-G, N-M2-C-H, N-M2-C-HN, N-P-C-F, N-P-C-G, N-P-C-H, N-P-C-HN,N-NS1-C-F, N-NS1-C-G, N-NS1-C-H, N-NS1-C-HN, N-NS2-C-F, N-NS2-C-G,N-NS2-C-H, N-NS2-C-HN, N-L-C-F, N-L-C-G, N-L-C-H, N-L-C-HN, M-N-C-F,M-N-C-G, M-N-C-H, M-N-C-HN, M-M-C-F, M-M-C-G, M-M-C-H, M-M-C-HN,M-M2-C-F, M-M2-C-G, M-M2-C-H, M-M2-C-HN, M-P-C-F, M-P-C-G, M-P-C-H,M-P-C-HN, M-NS1-C-F, M-NS1-C-G, M-NS1-C-H, M-NS1-C-HN, M-NS2-C-F,M-NS2-C-G, M-NS2-C-H, M-NS2-C-HN, M-L-C-F, M-L-C-G, M-L-C-H, M-L-C-HN,M2-N-C-F, M2-N-C-G, M2-N-C-H, M2-N-C-HN, M2-M-C-F, M2-M-C-G, M2-M-C-H,M2-M-C-HN, M2-M2-C-F, M2-M2-C-G, M2-M2-C-H, M2-M2-C-HN, M2-P-C-F,M2-P-C-G, M2-P-C-H, M2-P-C-HN, M2-NS1-C-F, M2-NS1-C-G, M2-NS1-C-H,M2-NS1-C-HN, M2-NS2-C-F, M2-NS2-C-G, M2-NS2-C-H, M2-NS2-C-HN, M2-L-C-F,M2-L-C-G, M2-L-C-H, M2-L-C-HN, P-N-C-F, P-N-C-G, P-N-C-H, P-N-C-HN,P-M-C-F, P-M-C-G, P-M-C-H, P-M-C-HN, P-M2-C-F, P-M2-C-G, P-M2-C-H,P-M2-C-HN, P-P-C-F, P-P-C-G, P-P-C-H, P-P-C-HN, P-NS1-C-F, P-NS1-C-G,P-NS1-C-H, P-NS1-C-HN, P-NS2-C-F, P-NS2-C-G, P-NS2-C-H, P-NS2-C-HN,P-L-C-F, P-L-C-G, P-L-C-H, P-L-C-HN, NS1-N-C-F, NS1-N-C-G, NS1-N-C-H,NS1-N-C-HN, NS1-M-C-F, NS1-M-C-G, NS1-M-C-H, NS1-M-C-HN, NS1-M2-C-F,NS1-M2-C-G, NS1-M2-C-H, NS1-M2-C-HN, NS1-P-C-F, NS1-P-C-G, NS1-P-C-H,NS1-P-C-HN, NS1-NS1-C-F, NS1-NS1-C-G, NS1-NS1-C-H, NS1-NS1-C-HN,NS1-NS2-C-F, NS1-NS2-C-G, NS1-NS2-C-H, NS1-NS2-C-HN, NS1-L-C-F,NS1-L-C-G, NS1-L-C-H, NS1-L-C-HN, NS2-N-C-F, NS2-N-C-G, NS2-N-C-H,NS2-N-C-HN, NS2-M-C-F, NS2-M-C-G, NS2-M-C-H, NS2-M-C-HN, NS2-M2-C-F,NS2-M2-C-G, NS2-M2-C-H, NS2-M2-C-HN, NS2-P-C-F, NS2-P-C-G, NS2-P-C-H,NS2-P-C-HN, NS2-NS1-C-F, NS2-NS1-C-G, NS2-NS1-C-H, NS2-NS1-C-HN,NS2-NS2-C-F, NS2-NS2-C-G, NS2-NS2-C-H, NS2-NS2-C-HN, NS2-L-C-F,NS2-L-C-G, NS2-L-C-H, NS2-L-C-HN, L-N-C-F, L-N-C-G, L-N-C-H, L-N-C-HN,L-M-C-F, L-M-C-G, L-M-C-H, L-M-C-HN, L-M2-C-F, L-M2-C-G, L-M2-C-H,L-M2-C-HN, L-L-C-F, L-P-C-G, L-P-C-H, L-P-C-HN, L-NS1-C-F, L-NS1-C-G,L-NS1-C-H, L-NS1-C-HN, L-NS2-C-F, L-NS2-C-G, L-NS2-C-H, L-NS2-C-HN,L-L-C-F, L-L-C-G, L-L-C-H, L-L-C-HN, N-C-N-K-F, N-C-N-K-G, N-C-N-K-H,N-C-N-K-HN, N-C-M-K-F, N-C-M-K-G, N-C-M-K-H, N-C-M-K-HN, N-C-M2-K-F,N-C-M2-K-G, N-C-M2-K-H, N-C-M2-K-HN, N-C-P-K-F, N-C-P-K-G, N-C-P-K-H,N-C-P-K-HN, N-C-NS1-K-F, N-C-NS1-K-G, N-C-NS1-K-H, N-C-NS1-K-HN,N-C-NS2-K-F, N-C-NS2-K-G, N-C-NS2-K-H, N-C-NS2-K-HN, N-C-L-K-F,N-C-L-K-G, N-C-L-K-H, N-C-L-K-HN, M-C-N-K-F, M-C-N-K-G, M-C-N-K-H,M-C-N-K-HN, M-C-M-K-F, M-C-M-K-G, M-C-M-K-H, M-C-M-K-HN, M-C-M2-K-F,M-C-M2-K-G, M-C-M2-K-H, M-C-M2-K-HN, M-C-P-K-F, M-C-P-K-G, M-C-P-K-H,M-C-P-K-HN, M-C-NS1-K-F, M-C-NS1-K-G, M-C-NS1-K-H, M-C-NS1-K-HN,M-C-NS2-K-F, M-C-NS2-K-G, M-C-NS2-K-H, M-C-NS2-K-HN, M-C-L-K-F,M-C-L-K-G, M-C-L-K-H, M-C-L-K-HN, M2-C-N-K-F, M2-C-N-K-G, M2-C-N-K-H,M2-C-N-K-HN, M2-C-M-K-F, M2-C-M-K-G, M2-C-M-K-H, M2-C-M-K-HN,M2-C-M2-K-F, M2-C-M2-K-G, M2-C-M2-K-H, M2-C-M2-K-HN, M2-C-P-K-F,M2-C-P-K-G, M2-C-P-K-H, M2-C-P-K-HN, M2-C-NS1-K-F, M2-C-NS1-K-G,M2-C-NS1-K-H, M2-C-NS1-K-HN, M2-C-NS2-K-F, M2-C-NS2-K-G, M2-C-NS2-K-H,M2-C-NS2-K-HN, M2-C-L-K-F, M2-C-L-K-G, M2-C-L-K-H, M2-C-L-K-HN,P-C-N-K-F, P-C-N-K-G, P-C-N-K-H, P-C-N-K-HN, P-C-M-K-F, P-C-M-K-G,P-C-M-K-H, P-C-M-K-HN, P-C-M2-K-F, P-C-M2-K-G, P-C-M2-K-H, P-C-M2-K-HN,P-C-P-K-F, P-C-P-K-G, P-C-P-K-H, P-C-P-K-HN, P-C-NS1-K-F, P-C-NS1-K-G,P-C-NS1-K-H, P-C-NS1-K-HN, P-C-NS2-K-F, P-C-NS2-K-G, P-C-NS2-K-H,P-C-NS2-K-HN, P-C-L-K-F, P-C-L-K-G, P-C-L-K-H, P-C-L-K-HN, NS1-C-N-K-F,NS1-C-N-K-G, NS1-C-N-K-H, NS1-C-N-K-HN, NS1-C-M-K-F, NS1-C-M-K-G,NS1-C-M-K-H, NS1-C-M-K-HN, NS1-C-M2-K-F, NS1-C-M2-K-G, NS1-C-M2-K-H,NS1-C-M2-K-HN, NS1-C-P-K-F, NS1-C-P-K-G, NS1-C-P-K-H, NS1-C-P-K-HN,NS1-C-NS1-K-F, NS1-C-NS1-K-G, NS1-C-NS1-K-H, NS1-C-NS1-K-HN,NS1-C-NS2-K-F, NS1-C-NS2-K-G, NS1-C-NS2-K-H, NS1-C-NS2-K-HN,NS1-C-L-K-F, NS1-C-L-K-G, NS1-C-L-K-H, NS1-C-L-K-HN, NS2-C-N-K-F,NS2-C-N-K-G, NS2-C-N-K-H, NS2-C-N-K-HN, NS2-C-M-K-F, NS2-C-M-K-G,NS2-C-M-K-H, NS2-C-M-K-HN, NS2-C-M2-K-F, NS2-C-M2-K-G, NS2-C-M2-K-H,NS2-C-M2-K-HN, NS2-C-P-K-F, NS2-C-P-K-G, NS2-C-P-K-H, NS2-C-P-K-HN,NS2-C-NS1-K-F, NS2-C-NS1-K-G, NS2-C-NS1-K-H, NS2-C-NS1-K-HN,NS2-C-NS2-K-F, NS2-C-NS2-K-G, NS2-C-NS2-K-H, NS2-C-NS2-K-HN,NS2-C-L-K-F, NS2-C-L-K-G, NS2-C-L-K-H, NS2-C-L-K-HN, L-C-N-K-F,L-C-N-K-G, L-C-N-K-H, L-C-N-K-HN, L-C-M-K-F, L-C-M-K-G, L-C-M-K-H,L-C-M-K-HN, L-C-M2-K-F, L-C-M2-K-G, L-C-M2-K-H, L-C-M2-K-HN, L-C-P-K-F,L-C-P-K-G, L-C-P-K-H, L-C-P-K-HN, L-C-NS1-K-F, L-C-NS1-K-G, L-C-NS1-K-H,L-C-NS1-K-HN, L-C-NS2-K-F, L-C-NS2-K-G, L-C-NS2-K-H, L-C-NS2-K-HN,L-C-L-K-F, L-C-L-K-G, L-C-L-K-H, or L-C-L-K-HN. Most preferably thearrangement is F-C-N-K-M2.

In preferred embodiments, the expression system is for use in theprophylaxis or treatment of viral infection, particularly preferably foruse in the prophylaxis or treatment of a paramyxovirus infection,preferably a RSV infection and/or in the manufacturing of medicament foruse in the prophylaxis or treatment of a paramyxovirus infection,preferably a RSV infection, and/or for use in methods of prophylaxis ortreatment of an of a paramyxovirus infection, preferably a RSVinfection.

In preferred embodiments, the expression system is for use in enhancingan immune response, preferably a B cell immune response against aparamyxovirus infection, preferably a RSV infection.

According to a preferred embodiment of the first aspect, the firstpolynucleotide encodes a viral protein of a orthomyxovirus or variantthereof which induces a reaction of the immune system (i.e. immuneresponse) in a host which is mediated by T cells, and the secondpolynucleotide encodes a viral protein of a orthomyxovirus or variantthereof that induces an anti-pathogenic B cell response against theorthomyxovirus. It is preferred that the orthomyxovirus whose viralproteins are encoded for by the first and second polynucleotide isselected from the genus of Influenzavirus A, Influenzavirus B,Influenzavirus C, Thogotoviris and Isavirus. In even more preferredembodiments, the orthomxyovirus is Influenzavirus A, preferably selectedfrom the subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3,H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, H10N7. According to apreferred embodiment of the first aspect, the second polynucleotideencodes a viral protein of an orthomyxovirus or variant thereof thatinduces an anti-pathogenic B cell response against the orthomyxovirus.

According to preferred embodiments of the first aspect firstpolynucleotide encodes a viral protein of a orthomyxovirus or variantthereof which induces a reaction of the immune system (i.e. immuneresponse) in a host which is mediated by T cells. A T cell responseinvolves the activation of antigen-specific T lymphocyte such as but notlimited to cytotoxic T cells (CTLs), T helper cells (T_(H) cells),central memory T cells (TCM cells), effector memory T cells (TEM cells),and regulatory T cells (Treg cells). A T cell response against a proteinis induced, if peptides of the protein are processed within the cell andpresented to T cells on the surface of the cell via the MHC I or MHC IIpathway. Thus, in the context of the present invention preferably thoseviral proteins or parts thereof are used for inducing a T cell responsethat are normally not exposed on the outside of the virus, e.g. nonstructural or internal proteins or parts of structural or surfaceproteins not accessible to B-cells on the outside of the virus.

According to a preferred embodiment of the first aspect, the secondpolynucleotide encodes a viral protein of an orthomyxovirus or variantthereof that induces an anti-pathogenic B cell response. A B cellresponse is an immune response based on the activation of B lymphocytes,which produce and secrete antigen specific antibodies. B cells involvedin such immune response include but are not limited to plasma B cells,memory B cells and B-1 cells. Thus, in the context of the presentinvention preferably those viral proteins or parts thereof are used forinducing a B cell response that are exposed on the outside of the virus,e.g. structural and/or surface proteins or at least those parts ofstructural and/or surface proteins accessible to B-cells on the outsideof the virus.

In a preferred embodiment of the first aspect the viral protein of anorthomyxovirus, which induces a T cell response is a non-structuraland/or internal protein of an orthomyxovirus, and/or the viral proteinof a orthomyxovirus, which induces an anti-pathogenic B cell response isa structural and/or surface protein of a orthomyxovirus.

It is preferred that the amino acid sequence of the structural (surface)and/or non-structural and/or internal protein comprises consecutivesegments or a consensus sequence of one or more different orthomyxovirusisolates.

In preferred embodiments, the structural protein is a protein exposed onthe surface of the native orthomyxovirus or a variant thereof. It ispreferred that the structural and/or surface protein triggers a T-cellindependent immune response such as but not limited to an antibodymediated immune response or an activation of the complement system. In aparticularly preferred embodiment, the structural and/or surface proteininduces an antibody mediated immune response. Such antibody mediatedimmune response is based on the activation of B cells which produce andsecrete antigen specific antibodies. B cells involved in such immuneresponse include but are not limited to plasma B cells, memory B cellsand B-1 cells.

In a further preferred embodiment, the membrane attachment domain of theprotein exposed on the surface of the native orthomyxovirus or variantthereof is functionally deleted, thus, either being structurally deletedor structurally present but not fulfilling its biological function. In aparticularly preferred embodiment, the amino acid sequence correspondingto the membrane attachment domain is deleted. The deletion of themembrane attachment region serves the purpose of ascertaining that theanti-pathogenic B cell response inducing protein is secreted from thecell into which the expression system of the invention has beenintroduced.

It is further preferred that the viral surface proteins of the nativeorthomyxovirus is selected from the group consisting of hemagglutinin(HA) and neuraminidase (NA). It is more preferred that the viral surfaceprotein of the native orthomyxovirus is hemagglutinin (HA).

In a preferred embodiment of the first aspect, HA comprises, essentiallyconsists of or consists of an amino acid sequence of HA of one influenzaA virus isolate or a consensus amino acid sequence of two or moredifferent influenza A virus isolates, preferably according to SEQ ID NO:8 or SEQ ID NO: 20, more preferably according to SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 21 or a variant of one of these sequences.

In preferred embodiments of the first aspect, the non-structural proteinis a conserved internal protein of orthomyxoviruses suitable forinducing a T cell mediated immune response against the paramyxovirus,involving the activation of antigen-specific T lymphocyte such as butnot limited to cytotoxic T cells (CTLs), T helper cells (T_(H) cells),central memory T cells (TCM cells), effector memory T cells (TEM cells),and regulatory T cells (Treg cells). Thus, preferably the T cellinducing protein of the orthomyxovirus does not comprise a secretionsignal.

Preferably, the non-structural and/or internal protein is selected fromthe group consisting of nucleoprotein NP, Matrix proteins M1 and M2, nonstructural proteins NS1 and NS2/NEP, and the RNA polymerases PA, PB1,PB2 and the protein PB1-F2 (PB1F2).

The nucleoprotein NP is a structural protein which encapsidates thenegative strand viral RNA. NP is one of the main determinants of speciesspecificity.

The protein M1 is a matrix protein of the influenza virus. It forms acoat inside the viral envelope. The M1 protein binds to the viral RNA.It also has multiple regulatory functions, performed by interaction withthe components of the host cell. The mechanisms regulated include a rolein the export of the viral ribonucleoproteins from the host cellnucleus, inhibition of viral transcription, and a role in the virusassembly and budding. The M1 protein forms a layer under the patches ofhost cell membrane that are rich with the viral hemagglutinin,neuraminidase and M2 transmembrane proteins, and facilitates budding ofthe mature viruses.

The non-structural NS1 protein is created by the internal proteinencoding, linear negative-sense, single stranded RNA, NS gene segmentand which also codes for the nuclear export protein or NEP, formerlyreferred to as the NS2 protein, which mediates the export of vRNPs. NS1also binds dsRNA. As a consequence of its binding to dsRNA, the NS1protein blocks the activation of the dsRNA-activated protein kinase(PKR) in vitro. This kinase phosphorylates the alpha subunit ofeukaryotic translation initiation factor 2 (elF-2 alpha), leading to adecrease in the rate of initiation of translation. In the absence ofNS1, this pathway is inhibited during anti-viral response to halt allprotein translation—thus stopping the synthesis of viral proteins;however, the influenza virus' NS1 protein is an agent that circumventshost defenses to allow viral gene transcription to occur.

In preferred embodiments, HA comprises an amino acid sequence of HA ofone influenza A virus isolate or a consensus amino acid sequence of twoor more different influenza A virus isolates, preferably according toSEQ ID NO: 9 or SEQ ID NO: 21, NP comprises an amino acid sequence of NPof one influenza A virus isolate or a consensus amino acid sequence oftwo or more different influenza A virus isolates, preferably accordingto SEQ ID NO: 11, and/or M1 comprises an amino acid sequence of M1 ofone influenza A virus isolate or a consensus amino acid sequence of twoor more different influenza A virus isolates, preferably according toSEQ ID NO: 12. It is further preferred that when NP comprises the aminoacid sequence according to SEQ ID NO: 11 and M1 comprises the amino acidsequence according to SEQ ID NO: 12.

In the context of the present invention, the structural and/or surfaceprotein encoded by the first polynucleotide is located either N- orC-terminally with respect to the non-structural and/or internal proteinencoded by the second polynucleotide. In a preferred embodiment, thenon-structural and/or internal protein encoded by the secondpolynucleotide is located C-terminally with respect to the structuraland/or surface protein encoded by the first polynucleotide.

More specifically, HA or NA can be located N- or C-terminally of NP, M1,M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2). In a preferredembodiment M1 is located N-terminally of HA.

Accordingly, embodiments of the present invention have the formula X-Yor Y-X, wherein “X” depicts HA or NA, preferably HA, and “Y” depicts NP,M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2), preferably NP orM1, and a “dash” depicts a peptide bond. Preferred arrangements are thefollowing:

HA-NP, HA-M1, HA-M2, HA-NS1, HA-NS2/NEP, HA-PA, HA-PB1, HA-PB2,HA-PB1F2, NP-HA, M1-HA, M2-HA, NS1-HA, NS2/NEP-HA, PA-HA, PB1-HA,PB2-HA, PB1F2-HA, NA-NP, NA-M1, NA-M2, NA-NS1, NA-NS2/NEP, NA-PA,NA-PB1, NA-PB2, NA-PB1F2, NP-NA, M1-NA, M2-NA, NS1-NA, NS2/NEP-NA,PA-NA, PB1-NA, PB2-NA or PB1F2-NA. A particularly preferred arrangementis M1-HA.

It is within the scope of the present invention that every protein canbe combined with any other protein.

In preferred embodiments of the first aspect, a polynucleotide encodinga cleavage site is positioned between the first polynucleotide and thesecond polynucleotide.

It is preferred that this cleavage site is either a self-cleaving site(i.e. a cleavage site within the amino acid sequence where this sequenceis cleaved or is cleavable without such cleavage involving anyadditional molecule or where the peptide-bond formation in this sequenceis prevented in the first place) or an endopeptidase cleavage site (i.e.a cleavage cite within the amino acid sequence where this sequence iscleaved or is cleavable by an endopeptidase, e.g. trypsin, pepsin,elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8,cathepsins). More preferably, the self-cleaving site is a 2A cleavagesite selected from the group consisting of a viral 2A peptide or 2A-likepeptide of Picornavirus, insect viruses, Aphtoviridae, Rotaviruses andTrypanosoma, preferably wherein the 2A cleavage site is the 2 A peptideof foot and mouth disease virus. Alternatively or additionally, thepolyprotein of the present invention can be cleaved by an autoprotease,i.e. a protease which cleaves peptide bonds in the same protein moleculewhich also comprises the protease. Examples of such autoproteases arethe NS2 protease from flaviviruses or the VP4 protease of birnaviruses.

In the context of the present invention, the cleavage site can bepositioned N-terminally with respect to the structural and/or surfaceprotein encoded by the first polynucleotide and C-terminally withrespect to the non-structural and/or internal protein encoded by thesecond polynucleotide. Alternatively the cleavage site can be positionedC-terminally with respect to the structural and/or surface proteinencoded by the first polynucleotide and N-terminally with respect to thenon-structural and/or internal protein encoded by the secondpolynucleotide. More specifically, the cleavage site can be positionedC- or N-terminally with respect to HA or NA and C- or N-terminally withrespect to NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2). Ina preferred embodiment the cleavage site is located C-terminally withrespect to NP M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2) andN-terminally with respect to HA or NA. It is particularly preferred thatthe cleavage site is located N-terminally with respect to HA andC-terminally with respect to M1.

Accordingly, embodiments of the present invention have the formula X-C-Yor Y-C-X, wherein “X” depicts HA or NA, preferably, HA and “Y” depictsNP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2), preferably NPor M1, “C” depicts a cleavage site, and a “dash” depicts a peptide bond.

Preferred arrangements are the following:

HA-C-NP, HA-C-M1, HA-C-M2, HA-C-NS1, HA-C-NS2/NEP, HA-C-PA, HA-C-PB1,HA-C-PB2, HA-C-PB1F2, NP-C-HA, M1-C-HA, M2-C-HA, NS1-C-HA, NS2/NEP-C-HA,PA-C-HA, PB1-C-HA, PB2-C-HA, PB1F2-C-HA, NA-C-NP, NA-C-M1, NA-C-M2,NA-C-NS1, NA-C-NS2/NEP, NA-C-PA, NA-C-PB1, NA-C-PB2, NA-C-PB1F2,NP-C-NA, M1-C-NA, M2-C-NA, NS1-C-NA, NS2/NEP-C-NA, PA-C-NA, PB1-C-NA,PB2-C-NA or PB1F2-C-NA. A particularly preferred arrangement is M1-C-HA.

It is within the scope of the present invention that every protein canbe combined with any other protein and that any two proteins can orcannot be connected or linked by a cleavage site.

In preferred embodiment of the first aspect, the expression systemfurther comprises a third polynucleotide encoding a non-structuraland/or internal protein of an orthomyxovirus or a variant thereof.Preferably, the non-structural and/or internal protein is of aorthomyxovirus selected from the genus of Influenzavirus A,Influenzavirus B, Influenzavirus C, Thogotoviris and Isavirus. In evenmore preferred embodiments, the orthomxyovirus is Influenzavirus A,preferably selected from the influenza A virus subtypes H1N1, H1N2,H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3,H7N4, H7N7, H9N2, H10N7, more preferably the influenza A virus subtypeH1N1.

In preferred embodiments the third polynucleotide is comprised on aseparate or on the same vector as the first polynucleotide and/or thesecond polynucleotide.

Accordingly, the first polynucleotide is comprised on one vector and thesecond polynucleotide is comprised on a second vector and the thirdpolynucleotide is comprised on a third vector. Alternatively oradditionally, the first and the second polynucleotide are comprised onthe same vector and the third polynucleotide is comprised on a separatevector, or the first and the third polynucleotide are comprised on thesame vector and the second polynucleotide is comprised on a separatevector, or the second and the third polynucleotide are comprised on thesame vector and the first polynucleotide is comprised on a separatevector. Alternatively or additionally, the first and the second and thethird polynucleotide are comprised on the same vector. It is preferredthat the first and the second and the third polynucleotide may becomprised on the same vector. It is particularly preferred that thefirst and the second and the third polynucleotide comprised on the samevector are linked in such that they are expressed as a viralpolyprotein. Preferably, the first and the second and the thirdpolynucleotide comprised on the same vector form an open reading frame.

It is further preferred that the non-structural and/or internal proteinencoded by the third polynucleotide is a conserved internal proteinsuitable for inducing a T cell mediated immune response against thevirus involving the activation of antigen-specific T lymphocyte such asbut not limited to cytotoxic T cells (CTLs), T helper cells (T_(H)cells), central memory T cells (TCM cells), effector memory T cells (TEMcells), and regulatory T cells (Treg cells).

Preferably, the non-structural and/or internal protein is selected fromthe group consisting of NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2(PB1F2), more preferably NP or M1.

It is preferred that the non-structural and/or internal protein encodedby the third polynucleotide differs from the non-structural and/orinternal protein encoded by the second polynucleotide.

The non-structural and/or internal proteins encoded by the second andthe third polynucleotide differ from each other in that they compriseamino acid sequences of different viral proteins. For instance, thismeans that the non-structural and/or internal protein encoded by thesecond polynucleotide comprises the amino acid sequence of the M1protein whilst the non-structural and/or internal protein encoded by thethird polynucleotide comprises the amino acid sequence of the NP proteinor vice versa.

The non-structural and/or internal protein encoded by the thirdpolynucleotide can be located either N- or C-terminally of thenon-structural and/or internal protein encoded by the secondpolynucleotide. In a preferred embodiment of the first aspect, thenon-structural and/or internal protein encoded by the thirdpolynucleotide is located C-terminally of the non-structural and/orinternal protein encoded by the second polynucleotide.

In preferred embodiments a polynucleotide encoding a linker ispositioned between the second polynucleotide and the thirdpolynucleotide. It is preferred that the linker is a flexible linker,preferably a flexible linker comprising an amino acid sequence accordingto SEQ ID NO: 6.

In embodiments of the first aspect, the protein encoded by the secondpolynucleotide is located N-terminally with respect to the proteinencoded by the first polynucleotide and/or the protein of the optionalthird polynucleotide, or the protein encoded by the secondpolynucleotide is located C-terminally with respect to the proteinencoded by the first polynucleotide and/or the protein of the optionalthird polynucleotide.

In even more preferred embodiments of this aspect, the firstpolynucleotide is located N-terminally with respect to the proteinencoded by the second polynucleotide and/or the protein of the optionalthird polynucleotide is located N-terminally with respect to the proteinencoded by the first polynucleotide or is located between the proteinencoded by the second polynucleotide and the protein encoded by thefirst polynucleotide; or the protein encoded by the first polynucleotideis located C-terminally with respect to the protein encoded by thesecond polynucleotide and/or the protein of the optional thirdpolynucleotide is located C-terminally with respect to the proteinencoded by the first polynucleotide or is located between the proteinencoded by the second polynucleotide and the protein encoded by thefirst polynucleotide. More specifically, HA, or NA are located C- orN-terminally with respect to NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 orPB1-F2 (PB1F2).

In a preferred embodiment HA is located C-terminally with respect to M1and NP is located N-terminally with respect to M1.

Accordingly, preferred embodiments of the present invention have theformula X-K-Y, Y-K-X, X-K-Y-Y, Y-Y-K-X, X-Y-K-Y, Y-K-Y-X, X-K-Y-K-Y,Y-K-Y-K-X, X-C-Y, Y-C-X, X-C-Y-Y, Y-Y-C-X, X-Y-C-Y, Y-C-Y-X, X-C-Y-C-Y,Y-C-Y-C-X, X-K-Y-C-Y, Y-C-Y-K-X, X-C-Y-K-Y, or Y-K-Y-C-X, wherein “X”depicts HA, or NA, preferably HA, and “Y” depicts NP, M1, M2, NS1,NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2), preferably NP or M1, “K”indicates that one or more peptide linkers are present in this position,“C” indicates that one or more cleavage sites are present in thisposition and a “dash” depicts a peptide bond. Preferred arrangements areY-K-Y-C-X. Even more preferred arrangements are the following:

HA-K-NP, NP-K-HA, HA-K-NP-NP, NP-NP-K-HA, HA-NP-K-NP, NP-K-NP-HA,HA-K-NP-K-NP, NP-K-NP-K-HA, HA-C-NP, NP-C-HA, HA-C-NP-NP, NP-NP-C-HA,HA-NP-C-NP, NP-C-NP-HA, HA-C-NP-C-NP, NP-C-NP-C-HA, HA-K-NP-C-NP,NP-C-NP-K-HA, HA-C-NP-K-NP, NP-K-NP-C-HA, HA-K-NP-M1, HA-K-M1-NP,NP-M1-K-HA, M1-NP-K-HA, HA-NP-K-M1, HA-M1-K-NP, NP-K-M1-HA, M1-K-NP-HA,HA-K-NP-K-M1, HA-K-M1-K-NP, NP-K-M1-K-HA, M1-K-NP-K-HA, HA-C-NP-M1,HA-C-M1-NP, NP-M1-C-HA, M1-NP-C-HA, HA-NP-C-M1, HA-M1-C-NP, NP-C-M1-HA,M1-C-NP-HA, HA-C-NP-C-M1, HA-C-M1-C-NP, NP-C-M1-C-HA, M1-C-NP-C-HA,HA-K-NP-C-M1, HA-K-M1-C-NP, NP-C-M1-K-HA, M1-C-NP-K-HA, HA-C-NP-K-M1,HA-C-M1-K-NP, NP-K-M1-C-HA, M1-K-NP-C-HA, HA-K-NP-M2, HA-K-M2-NP,NP-M2-K-HA, M2-NP-K-HA, HA-NP-K-M2, HA-M2-K-NP, NP-K-M2-HA, M2-K-NP-HA,HA-K-NP-K-M2, HA-K-M2-K-NP, NP-K-M2-K-HA, M2-K-NP-K-HA, HA-C-NP-M2,HA-C-M2-NP, NP-M2-C-HA, M2-NP-C-HA, HA-NP-C-M2, HA-M2-C-NP, NP-C-M2-HA,M2-C-NP-HA, HA-C-NP-C-M2, HA-C-M2-C-NP, NP-C-M2-C-HA, M2-C-NP-C-HA,HA-K-NP-C-M2, HA-K-M2-C-NP, NP-C-M2-K-HA, M2-C-NP-K-HA, HA-C-NP-K-M2,HA-C-M2-K-NP, NP-K-M2-C-HA, M2-K-NP-C-HA, HA-K-NP-NS1, HA-K-NS1-NP,NP-NS1-K-HA, NS1-NP-K-HA, HA-NP-K-NS1, HA-NS1-K-NP, NP-K-NS1-HA,NS1-K-NP-HA, HA-K-NP-K-NS1, HA-K-NS1-K-NP, NP-K-NS1-K-HA, NS1-K-NP-K-HA,HA-C-NP-NS1, HA-C-NS1-NP, NP-NS1-C-HA, NS1-NP-C-HA, HA-NP-C-NS1,HA-NS1-C-NP, NP-C-NS1-HA, NS1-C-NP-HA, HA-C-NP-C-NS1, HA-C-NS1-C-NP,NP-C-NS1-C-HA, NS1-C-NP-C-HA, HA-K-NP-C-NS1, HA-K-NS1-C-NP,NP-C-NS1-K-HA, NS1-C-NP-K-HA, HA-C-NP-K-NS1, HA-C-NS1-K-NP,NP-K-NS1-C-HA, NS1-K-NP-C-HA, HA-K-NP-NS2/NEP, HA-K-NS2/NEP-NP,NP-NS2/NEP-K-HA, NS2/NEP-NP-K-HA, HA-NP-K-NS2/NEP, HA-NS2/NEP-K-NP,NP-K-NS2/NEP-HA, NS2/NEP-K-NP-HA, HA-K-NP-K-NS2/NEP, HA-K-NS2/NEP-K-NP,NP-K-NS2/NEP-K-HA, NS2/NEP-K-NP-K-HA, HA-C-NP-NS2/NEP, HA-C-NS2/NEP-NP,NP-NS2/NEP-C-HA, NS2/NEP-NP-C-HA, HA-NP-C-NS2/NEP, HA-NS2/NEP-C-NP,NP-C-NS2/NEP-HA, NS2/NEP-C-NP-HA, HA-C-NP-C-NS2/NEP, HA-C-NS2/NEP-C-NP,NP-C-NS2/NEP-C-HA, NS2/NEP-C-NP-C-HA, HA-K-NP-C-NS2/NEP,HA-K-NS2/NEP-C-NP, NP-C-NS2/NEP-K-HA, NS2/NEP-C-NP-K-HA,HA-C-NP-K-NS2/NEP, HA-C-NS2/NEP-K-NP, NP-K-NS2/NEP-C-HA,NS2/NEP-K-NP-C-HA, HA-K-NP-PA, HA-K-PA-NP, NP-PA-K-HA, PA-NP-K-HA,HA-NP-K-PA, HA-PA-K-NP, NP-K-PA-HA, PA-K-NP-HA, HA-K-NP-K-PA,HA-K-PA-K-NP, NP-K-PA-K-HA, PA-K-NP-K-HA, HA-C-NP-PA, HA-C-PA-NP,NP-PA-C-HA, PA-NP-C-HA, HA-NP-C-PA, HA-PA-C-NP, NP-C-PA-HA, PA-C-NP-HA,HA-C-NP-C-PA, HA-C-PA-C-NP, NP-C-PA-C-HA, PA-C-NP-C-HA, HA-K-NP-C-PA,HA-K-PA-C-NP, NP-C-PA-K-HA, PA-C-NP-K-HA, HA-C-NP-K-PA, HA-C-PA-K-NP,NP-K-PA-C-HA, PA-K-NP-C-HA, HA-K-NP-PB1, HA-K-PB1-NP, NP-PB1-K-HA,PB1-NP-K-HA, HA-NP-K-PB1, HA-PB1-K-NP, NP-K-PB1-HA, PB1-K-NP-HA,HA-K-NP-K-PB1, HA-K-PB1-K-NP, NP-K-PB1-K-HA, PB1-K-NP-K-HA, HA-C-NP-PB1,HA-C-PB1-NP, NP-PB1-C-HA, PB1-NP-C-HA, HA-NP-C-PB1, HA-PB1-C-NP,NP-C-PB1-HA, PB1-C-NP-HA, HA-C-NP-C-PB1, HA-C-PB1-C-NP, NP-C-PB1-C-HA,PB1-C-NP-C-HA, HA-K-NP-C-PB1, HA-K-PB1-C-NP, NP-C-PB1-K-HA,PB1-C-NP-K-HA, HA-C-NP-K-PB1, HA-C-PB1-K-NP, NP-K-PB1-C-HA,PB1-K-NP-C-HA, HA-K-NP-PB2, HA-K-PB2-NP, NP-PB2-K-HA, PB2-NP-K-HA,HA-NP-K-PB2, HA-PB2-K-NP, NP-K-PB2-HA, PB2-K-NP-HA, HA-K-NP-K-PB2,HA-K-PB2-K-NP, NP-K-PB2-K-HA, PB2-K-NP-K-HA, HA-C-NP-PB2, HA-C-PB2-NP,NP-PB2-C-HA, PB2-NP-C-HA, HA-NP-C-PB2, HA-PB2-C-NP, NP-C-PB2-HA,PB2-C-NP-HA, HA-C-NP-C-PB2, HA-C-PB2-C-NP, NP-C-PB2-C-HA, PB2-C-NP-C-HA,HA-K-NP-C-PB2, HA-K-PB2-C-NP, NP-C-PB2-K-HA, PB2-C-NP-K-HA,HA-C-NP-K-PB2, HA-C-PB2-K-NP, NP-K-PB2-C-HA, PB2-K-NP-C-HA,HA-K-NP-PB1F2, HA-K-PB1F2-NP, NP-PB1F2-K-HA, PB1F2-NP-K-HA,HA-NP-K-PB1F2, HA-PB1F2-K-NP, NP-K-PB1F2-HA, PB1F2-K-NP-HA,HA-K-NP-K-PB1F2, HA-K-PB1F2-K-NP, NP-K-PB1F2-K-HA, PB1F2-K-NP-K-HA,HA-C-NP-PB1F2, HA-C-PB1F2-NP, NP-PB1F2-C-HA, PB1F2-NP-C-HA,HA-NP-C-PB1F2, HA-PB1F2-C-NP, NP-C-PB1F2-HA, PB1F2-C-NP-HA,HA-C-NP-C-PB1F2, HA-C-PB1F2-C-NP, NP-C-PB1F2-C-HA, PB1F2-C-NP-C-HA,HA-K-NP-C-PB1F2, HA-K-PB1F2-C-NP, NP-C-PB1F2-K-HA, PB1F2-C-NP-K-HA,HA-C-NP-K-PB1F2, HA-C-PB1F2-K-NP, NP-K-PB1F2-C-HA, PB1F2-K-NP-C-HA,HA-K-M1, M1-K-HA, HA-K-M1-M1, M1-M1-K-HA, HA-M1-K-M1, M1-K-M1-HA,HA-K-M1-K-M1, M1-K-M1-K-HA, HA-C-M1, M1-C-HA, HA-C-M1-M1, M1-M1-C-HA,HA-M1-C-M1, M1-C-M1-HA, HA-C-M1-C-M1, M1-C-M1-C-HA, HA-K-M1-C-M1,M1-C-M1-K-HA, HA-C-M1-K-M1, M1-K-M1-C-HA, HA-K-M1-M2, HA-K-M2-M1,M1-M2-K-HA, M2-M1-K-HA, HA-M1-K-M2, HA-M2-K-M1, M1-K-M2-HA, M2-K-M1-HA,HA-K-M1-K-M2, HA-K-M2-K-M1, M1-K-M2-K-HA, M2-K-M1-K-HA, HA-C-M1-M2,HA-C-M2-M1, M1-M2-C-HA, M2-M1-C-HA, HA-M1-C-M2, HA-M2-C-M1, M1-C-M2-HA,M2-C-M1-HA, HA-C-M1-C-M2, HA-C-M2-C-M1, M1-C-M2-C-HA, M2-C-M1-C-HA,HA-K-M1-C-M2, HA-K-M2-C-M1, M1-C-M2-K-HA, M2-C-M1-K-HA, HA-C-M1-K-M2,HA-C-M2-K-M1, M1-K-M2-C-HA, M2-K-M1-C-HA, HA-K-M1-NS1, HA-K-NS1-M1,M1-NS1-K-HA, NS1-M1-K-HA, HA-M1-K-NS1, HA-NS1-K-M1, M1-K-NS1-HA,NS1-K-M1-HA, HA-K-M1-K-NS1, HA-K-NS1-K-M1, M1-K-NS1-K-HA, NS1-K-M1-K-HA,HA-C-M1-NS1, HA-C-NS1-M1, M1-NS1-C-HA, NS1-M1-C-HA, HA-M1-C-NS1,HA-NS1-C-M1, M1-C-NS1-HA, NS1-C-M1-HA, HA-C-M1-C-NS1, HA-C-NS1-C-M1,M1-C-NS1-C-HA, NS1-C-M1-C-HA, HA-K-M1-C-NS1, HA-K-NS1-C-M1,M1-C-NS1-K-HA, NS1-C-M1-K-HA, HA-C-M1-K-NS1, HA-C-NS1-K-M1,M1-K-NS1-C-HA, NS1-K-M1-C-HA, HA-K-M1-NS2/NEP, HA-K-NS2/NEP-M1,M1-NS2/NEP-K-HA, NS2/NEP-M1-K-HA, HA-M1-K-NS2/NEP, HA-NS2/NEP-K-M1,M1-K-NS2/NEP-HA, NS2/NEP-K-M1-HA, HA-K-M1-K-NS2/NEP, HA-K-NS2/NEP-K-M1,M1-K-NS2/NEP-K-HA, NS2/NEP-K-M1-K-HA, HA-C-M1-NS2/NEP, HA-C-NS2/NEP-M1,M1-NS2/NEP-C-HA, NS2/NEP-M1-C-HA, HA-M1-C-NS2/NEP, HA-NS2/NEP-C-M1,M1-C-NS2/NEP-HA, NS2/NEP-C-M1-HA, HA-C-M1-C-NS2/NEP, HA-C-NS2/NEP-C-M1,M1-C-NS2/NEP-C-HA, NS2/NEP-C-M1-C-HA, HA-K-M1-C-NS2/NEP,HA-K-NS2/NEP-C-M1, M1-C-NS2/NEP-K-HA, NS2/NEP-C-M1-K-HA,HA-C-M1-K-NS2/NEP, HA-C-NS2/NEP-K-M1, M1-K-NS2/NEP-C-HA,NS2/NEP-K-M1-C-HA, HA-K-M1-PA, HA-K-PA-M1, M1-PA-K-HA, PA-M1-K-HA,HA-M1-K-PA, HA-PA-K-M1, M1-K-PA-HA, PA-K-M1-HA, HA-K-M1-K-PA,HA-K-PA-K-M1, M1-K-PA-K-HA, PA-K-M1-K-HA, HA-C-M1-PA, HA-C-PA-M1,M1-PA-C-HA, PA-M1-C-HA, HA-M1-C-PA, HA-PA-C-M1, M1-C-PA-HA, PA-C-M1-HA,HA-C-M1-C-PA, HA-C-PA-C-M1, M1-C-PA-C-HA, PA-C-M1-C-HA, HA-K-M1-C-PA,HA-K-PA-C-M1, M1-C-PA-K-HA, PA-C-M1-K-HA, HA-C-M1-K-PA, HA-C-PA-K-M1,M1-K-PA-C-HA, PA-K-M1-C-HA, HA-K-M1-PB1, HA-K-PB1-M1, M1-PB1-K-HA,PB1-M1-K-HA, HA-M1-K-PB1, HA-PB1-K-M1, M1-K-PB1-HA, PB1-K-M1-HA,HA-K-M1-K-PB1, HA-K-PB1-K-M1, M1-K-PB1-K-HA, PB1-K-M1-K-HA, HA-C-M1-PB1,HA-C-PB1-M1, M1-PB1-C-HA, PB1-M1-C-HA, HA-M1-C-PB1, HA-PB1-C-M1,M1-C-PB1-HA, PB1-C-M1-HA, HA-C-M1-C-PB1, HA-C-PB1-C-M1, M1-C-PB1-C-HA,PB1-C-M1-C-HA, HA-K-M1-C-PB1, HA-K-PB1-C-M1, M1-C-PB1-K-HA,PB1-C-M1-K-HA, HA-C-M1-K-PB1, HA-C-PB1-K-M1, M1-K-PB1-C-HA,PB1-K-M1-C-HA, HA-K-M1-PB2, HA-K-PB2-M1, M1-PB2-K-HA, PB2-M1-K-HA,HA-M1-K-PB2, HA-PB2-K-M1, M1-K-PB2-HA, PB2-K-M1-HA, HA-K-M1-K-PB2,HA-K-PB2-K-M1, M1-K-PB2-K-HA, PB2-K-M1-K-HA, HA-C-M1-PB2, HA-C-PB2-M1,M1-PB2-C-HA, PB2-M1-C-HA, HA-M1-C-PB2, HA-PB2-C-M1, M1-C-PB2-HA,PB2-C-M1-HA, HA-C-M1-C-PB2, HA-C-PB2-C-M1, M1-C-PB2-C-HA, PB2-C-M1-C-HA,HA-K-M1-C-PB2, HA-K-PB2-C-M1, M1-C-PB2-K-HA, PB2-C-M1-K-HA,HA-C-M1-K-PB2, HA-C-PB2-K-M1, M1-K-PB2-C-HA, PB2-K-M1-C-HA,HA-K-M1-PB1F2, HA-K-PB1F2-M1, M1-PB1F2-K-HA, PB1F2-M1-K-HA,HA-M1-K-PB1F2, HA-PB1F2-K-M1, M1-K-PB1F2-HA, PB1F2-K-M1-HA,HA-K-M1-K-PB1F2, HA-K-PB1F2-K-M1, M1-K-PB1F2-K-HA, PB1F2-K-M1-K-HA,HA-C-M1-PB1F2, HA-C-PB1F2-M1, M1-PB1F2-C-HA, PB1F2-M1-C-HA,HA-M1-C-PB1F2, HA-PB1F2-C-M1, M1-C-PB1F2-HA, PB1F2-C-M1-HA,HA-C-M1-C-PB1F2, HA-C-PB1F2-C-M1, M1-C-PB1F2-C-HA, PB1F2-C-M1-C-HA,HA-K-M1-C-PB1F2, HA-K-PB1F2-C-M1, M1-C-PB1F2-K-HA, PB1F2-C-M1-K-HA,HA-C-M1-K-PB1F2, HA-C-PB1F2-K-M1, M1-K-PB1F2-C-HA, PB1F2-K-M1-C-HA,HA-K-M2, M2-K-HA, HA-K-M2-M2, M2-M2-K-HA, HA-M2-K-M2, M2-K-M2-HA,HA-K-M2-K-M2, M2-K-M2-K-HA, HA-C-M2, M2-C-HA, HA-C-M2-M2, M2-M2-C-HA,HA-M2-C-M2, M2-C-M2-HA, HA-C-M2-C-M2, M2-C-M2-C-HA, HA-K-M2-C-M2,M2-C-M2-K-HA, HA-C-M2-K-M2, M2-K-M2-C-HA, HA-K-M2-NS1, HA-K-NS1-M2,M2-NS1-K-HA, NS1-M2-K-HA, HA-M2-K-NS1, HA-NS1-K-M2, M2-K-NS1-HA,NS1-K-M2-HA, HA-K-M2-K-NS1, HA-K-NS1-K-M2, M2-K-NS1-K-HA, NS1-K-M2-K-HA,HA-C-M2-NS1, HA-C-NS1-M2, M2-NS1-C-HA, NS1-M2-C-HA, HA-M2-C-NS1,HA-NS1-C-M2, M2-C-NS1-HA, NS1-C-M2-HA, HA-C-M2-C-NS1, HA-C-NS1-C-M2,M2-C-NS1-C-HA, NS1-C-M2-C-HA, HA-K-M2-C-NS1, HA-K-NS1-C-M2,M2-C-NS1-K-HA, NS1-C-M2-K-HA, HA-C-M2-K-NS1, HA-C-NS1-K-M2,M2-K-NS1-C-HA, NS1-K-M2-C-HA, HA-K-M2-NS2/NEP, HA-K-NS2/NEP-M2,M2-NS2/NEP-K-HA, NS2/NEP-M2-K-HA, HA-M2-K-NS2/NEP, HA-NS2/NEP-K-M2,M2-K-NS2/NEP-HA, NS2/NEP-K-M2-HA, HA-K-M2-K-NS2/NEP, HA-K-NS2/NEP-K-M2,M2-K-NS2/NEP-K-HA, NS2/NEP-K-M2-K-HA, HA-C-M2-NS2/NEP, HA-C-NS2/NEP-M2,M2-NS2/NEP-C-HA, NS2/NEP-M2-C-HA, HA-M2-C-NS2/NEP, HA-NS2/NEP-C-M2,M2-C-NS2/NEP-HA, NS2/NEP-C-M2-HA, HA-C-M2-C-NS2/NEP, HA-C-NS2/NEP-C-M2,M2-C-NS2/NEP-C-HA, NS2/NEP-C-M2-C-HA, HA-K-M2-C-NS2/NEP,HA-K-NS2/NEP-C-M2, M2-C-NS2/NEP-K-HA, NS2/NEP-C-M2-K-HA,HA-C-M2-K-NS2/NEP, HA-C-NS2/NEP-K-M2, M2-K-NS2/NEP-C-HA,NS2/NEP-K-M2-C-HA, HA-K-M2-PA, HA-K-PA-M2, M2-PA-K-HA, PA-M2-K-HA,HA-M2-K-PA, HA-PA-K-M2, M2-K-PA-HA, PA-K-M2-HA, HA-K-M2-K-PA,HA-K-PA-K-M2, M2-K-PA-K-HA, PA-K-M2-K-HA, HA-C-M2-PA, HA-C-PA-M2,M2-PA-C-HA, PA-M2-C-HA, HA-M2-C-PA, HA-PA-C-M2, M2-C-PA-HA, PA-C-M2-HA,HA-C-M2-C-PA, HA-C-PA-C-M2, M2-C-PA-C-HA, PA-C-M2-C-HA, HA-K-M2-C-PA,HA-K-PA-C-M2, M2-C-PA-K-HA, PA-C-M2-K-HA, HA-C-M2-K-PA, HA-C-PA-K-M2,M2-K-PA-C-HA, PA-K-M2-C-HA, HA-K-M2-PB1, HA-K-PB1-M2, M2-PB1-K-HA,PB1-M2-K-HA, HA-M2-K-PB1, HA-PB1-K-M2, M2-K-PB1-HA, PB1-K-M2-HA,HA-K-M2-K-PB1, HA-K-PB1-K-M2, M2-K-PB1-K-HA, PB1-K-M2-K-HA, HA-C-M2-PB1,HA-C-PB1-M2, M2-PB1-C-HA, PB1-M2-C-HA, HA-M2-C-PB1, HA-PB1-C-M2,M2-C-PB1-HA, PB1-C-M2-HA, HA-C-M2-C-PB1, HA-C-PB1-C-M2, M2-C-PB1-C-HA,PB1-C-M2-C-HA, HA-K-M2-C-PB1, HA-K-PB1-C-M2, M2-C-PB1-K-HA,PB1-C-M2-K-HA, HA-C-M2-K-PB1, HA-C-PB1-K-M2, M2-K-PB1-C-HA,PB1-K-M2-C-HA, HA-K-M2-PB2, HA-K-PB2-M2, M2-PB2-K-HA, PB2-M2-K-HA,HA-M2-K-PB2, HA-PB2-K-M2, M2-K-PB2-HA, PB2-K-M2-HA, HA-K-M2-K-PB2,HA-K-PB2-K-M2, M2-K-PB2-K-HA, PB2-K-M2-K-HA, HA-C-M2-PB2, HA-C-PB2-M2,M2-PB2-C-HA, PB2-M2-C-HA, HA-M2-C-PB2, HA-PB2-C-M2, M2-C-PB2-HA,PB2-C-M2-HA, HA-C-M2-C-PB2, HA-C-PB2-C-M2, M2-C-PB2-C-HA, PB2-C-M2-C-HA,HA-K-M2-C-PB2, HA-K-PB2-C-M2, M2-C-PB2-K-HA, PB2-C-M2-K-HA,HA-C-M2-K-PB2, HA-C-PB2-K-M2, M2-K-PB2-C-HA, PB2-K-M2-C-HA,HA-K-M2-PB1F2, HA-K-PB1F2-M2, M2-PB1F2-K-HA, PB1F2-M2-K-HA,HA-M2-K-PB1F2, HA-PB1F2-K-M2, M2-K-PB1F2-HA, PB1F2-K-M2-HA,HA-K-M2-K-PB1F2, HA-K-PB1F2-K-M2, M2-K-PB1F2-K-HA, PB1F2-K-M2-K-HA,HA-C-M2-PB1F2, HA-C-PB1F2-M2, M2-PB1F2-C-HA, PB1F2-M2-C-HA,HA-M2-C-PB1F2, HA-PB1F2-C-M2, M2-C-PB1F2-HA, PB1F2-C-M2-HA,HA-C-M2-C-PB1F2, HA-C-PB1F2-C-M2, M2-C-PB1F2-C-HA, PB1F2-C-M2-C-HA,HA-K-M2-C-PB1F2, HA-K-PB1F2-C-M2, M2-C-PB1F2-K-HA, PB1F2-C-M2-K-HA,HA-C-M2-K-PB1F2, HA-C-PB1F2-K-M2, M2-K-PB1F2-C-HA, PB1F2-K-M2-C-HA,HA-K-NS1, NS1-K-HA, HA-K-NS1-NS1, NS1-NS1-K-HA, HA-NS1-K-NS1,NS1-K-NS1-HA, HA-K-NS1-K-NS1, NS1-K-NS1-K-HA, HA-C-NS1, NS1-C-HA,HA-C-NS1-NS1, NS1-NS1-C-HA, HA-NS1-C-NS1, NS1-C-NS1-HA, HA-C-NS1-C-NS1,NS1-C-NS1-C-HA, HA-K-NS1-C-NS1, NS1-C-NS1-K-HA, HA-C-NS1-K-NS1,NS1-K-NS1-C-HA, HA-K-NS1-NS2/NEP, HA-K-NS2/NEP-NS1, NS1-NS2/NEP-K-HA,NS2/NEP-NS1-K-HA, HA-NS1-K-NS2/NEP, HA-NS2/NEP-K-NS1, NS1-K-NS2/NEP-HA,NS2/NEP-K-NS1-HA, HA-K-NS1-K-NS2/NEP, HA-K-NS2/NEP-K-NS1,NS1-K-NS2/NEP-K-HA, NS2/NEP-K-NS1-K-HA, HA-C-NS1-NS2/NEP,HA-C-NS2/NEP-NS1, NS1-NS2/NEP-C-HA, NS2/NEP-NS1-C-HA, HA-NS1-C-NS2/NEP,HA-NS2/NEP-C-NS1, NS1-C-NS2/NEP-HA, NS2/NEP-C-NS1-HA,HA-C-NS1-C-NS2/NEP, HA-C-NS2/NEP-C-NS1, NS1-C-NS2/NEP-C-HA,NS2/NEP-C-NS1-C-HA, HA-K-NS1-C-NS2/NEP, HA-K-NS2/NEP-C-NS1,NS1-C-NS2/NEP-K-HA, NS2/NEP-C-NS1-K-HA, HA-C-NS1-K-NS2/NEP,HA-C-NS2/NEP-K-NS1, NS1-K-NS2/NEP-C-HA, NS2/NEP-K-NS1-C-HA, HA-K-NS1-PA,HA-K-PA-NS1, NS1-PA-K-HA, PA-NS1-K-HA, HA-NS1-K-PA, HA-PA-K-NS1,NS1-K-PA-HA, PA-K-NS1-HA, HA-K-NS1-K-PA, HA-K-PA-K-NS1, NS1-K-PA-K-HA,PA-K-NS1-K-HA, HA-C-NS1-PA, HA-C-PA-NS1, NS1-PA-C-HA, PA-NS1-C-HA,HA-NS1-C-PA, HA-PA-C-NS1, NS1-C-PA-HA, PA-C-NS1-HA, HA-C-NS1-C-PA,HA-C-PA-C-NS1, NS1-C-PA-C-HA, PA-C-NS1-C-HA, HA-K-NS1-C-PA,HA-K-PA-C-NS1, NS1-C-PA-K-HA, PA-C-NS1-K-HA, HA-C-NS1-K-PA,HA-C-PA-K-NS1, NS1-K-PA-C-HA, PA-K-NS1-C-HA, HA-K-NS1-PB1, HA-K-PB1-NS1,NS1-PB1-K-HA, PB1-NS1-K-HA, HA-NS1-K-PB1, HA-PB1-K-NS1, NS1-K-PB1-HA,PB1-K-NS1-HA, HA-K-NS1-K-PB1, HA-K-PB1-K-NS1, NS1-K-PB1-K-HA,PB1-K-NS1-K-HA, HA-C-NS1-PB1, HA-C-PB1-NS1, NS1-PB1-C-HA, PB1-NS1-C-HA,HA-NS1-C-PB1, HA-PB1-C-NS1, NS1-C-PB1-HA, PB1-C-NS1-HA, HA-C-NS1-C-PB1,HA-C-PB1-C-NS1, NS1-C-PB1-C-HA, PB1-C-NS1-C-HA, HA-K-NS1-C-PB1,HA-K-PB1-C-NS1, NS1-C-PB1-K-HA, PB1-C-NS1-K-HA, HA-C-NS1-K-PB1,HA-C-PB1-K-NS1, NS1-K-PB1-C-HA, PB1-K-NS1-C-HA, HA-K-NS1-PB2,HA-K-PB2-NS1, NS1-PB2-K-HA, PB2-NS1-K-HA, HA-NS1-K-PB2, HA-PB2-K-NS1,NS1-K-PB2-HA, PB2-K-NS1-HA, HA-K-NS1-K-PB2, HA-K-PB2-K-NS1,NS1-K-PB2-K-HA, PB2-K-NS1-K-HA, HA-C-NS1-PB2, HA-C-PB2-NS1,NS1-PB2-C-HA, PB2-NS1-C-HA, HA-NS1-C-PB2, HA-PB2-C-NS1, NS1-C-PB2-HA,PB2-C-NS1-HA, HA-C-NS1-C-PB2, HA-C-PB2-C-NS1, NS1-C-PB2-C-HA,PB2-C-NS1-C-HA, HA-K-NS1-C-PB2, HA-K-PB2-C-NS1, NS1-C-PB2-K-HA,PB2-C-NS1-K-HA, HA-C-NS1-K-PB2, HA-C-PB2-K-NS1, NS1-K-PB2-C-HA,PB2-K-NS1-C-HA, HA-K-NS1-PB1F2, HA-K-PB1F2-NS1, NS1-PB1F2-K-HA,PB1F2-NS1-K-HA, HA-NS1-K-PB1F2, HA-PB1F2-K-NS1, NS1-K-PB1F2-HA,PB1F2-K-NS1-HA, HA-K-NS1-K-PB1F2, HA-K-PB1F2-K-NS1, NS1-K-PB1F2-K-HA,PB1F2-K-NS1-K-HA, HA-C-NS1-PB1F2, HA-C-PB1F2-NS1, NS1-PB1F2-C-HA,PB1F2-NS1-C-HA, HA-NS1-C-PB1F2, HA-PB1F2-C-NS1, NS1-C-PB1F2-HA,PB1F2-C-NS1-HA, HA-C-NS1-C-PB1F2, HA-C-PB1F2-C-NS1, NS1-C-PB1F2-C-HA,PB1F2-C-NS1-C-HA, HA-K-NS1-C-PB1F2, HA-K-PB1F2-C-NS1, NS1-C-PB1F2-K-HA,PB1F2-C-NS1-K-HA, HA-C-NS1-K-PB1F2, HA-C-PB1F2-K-NS1, NS1-K-PB1F2-C-HA,PB1F2-K-NS1-C-HA, HA-K-NS2/NEP, NS2/NEP-K-HA, HA-K-NS2/NEP-NS2/NEP,NS2/NEP-NS2/NEP-K-HA, HA-NS2/NEP-K-NS2/NEP, NS2/NEP-K-NS2/NEP-HA,HA-K-NS2/NEP-K-NS2/NEP, NS2/NEP-K-NS2/NEP-K-HA, HA-C-NS2/NEP,NS2/NEP-C-HA, HA-C-NS2/NEP-NS2/NEP, NS2/NEP-NS2/NEP-C-HA,HA-NS2/NEP-C-NS2/NEP, NS2/NEP-C-NS2/NEP-HA, HA-C-NS2/NEP-C-NS2/NEP,NS2/NEP-C-NS2/NEP-C-HA, HA-K-NS2/NEP-C-NS2/NEP, NS2/NEP-C-NS2/NEP-K-HA,HA-C-NS2/NEP-K-NS2/NEP, NS2/NEP-K-NS2/NEP-C-HA, HA-K-NS2/NEP-PA,HA-K-PA-NS2/NEP, NS2/NEP-PA-K-HA, PA-NS2/NEP-K-HA, HA-NS2/NEP-K-PA,HA-PA-K-NS2/NEP, NS2/NEP-K-PA-HA, PA-K-NS2/NEP-HA, HA-K-NS2/NEP-K-PA,HA-K-PA-K-NS2/NEP, NS2/NEP-K-PA-K-HA, PA-K-NS2/NEP-K-HA,HA-C-NS2/NEP-PA, HA-C-PA-NS2/NEP, NS2/NEP-PA-C-HA, PA-NS2/NEP-C-HA,HA-NS2/NEP-C-PA, HA-PA-C-NS2/NEP, NS2/NEP-C-PA-HA, PA-C-NS2/NEP-HA,HA-C-NS2/NEP-C-PA, HA-C-PA-C-NS2/NEP, NS2/NEP-C-PA-C-HA,PA-C-NS2/NEP-C-HA, HA-K-NS2/NEP-C-PA, HA-K-PA-C-NS2/NEP,NS2/NEP-C-PA-K-HA, PA-C-NS2/NEP-K-HA, HA-C-NS2/NEP-K-PA,HA-C-PA-K-NS2/NEP, NS2/NEP-K-PA-C-HA, PA-K-NS2/NEP-C-HA,HA-K-NS2/NEP-PB1, HA-K-PB1-NS2/NEP, NS2/NEP-PB1-K-HA, PB1-NS2/NEP-K-HA,HA-NS2/NEP-K-PB1, HA-PB1-K-NS2/NEP, NS2/NEP-K-PB1-HA, PB1-K-NS2/NEP-HA,HA-K-NS2/NEP-K-PB1, HA-K-PB1-K-NS2/NEP, NS2/NEP-K-PB1-K-HA,PB1-K-NS2/NEP-K-HA, HA-C-NS2/NEP-PB1, HA-C-PB1-NS2/NEP,NS2/NEP-PB1-C-HA, PB1-NS2/NEP-C-HA, HA-NS2/NEP-C-PB1, HA-PB1-C-NS2/NEP,NS2/NEP-C-PB1-HA, PB1-C-NS2/NEP-HA, HA-C-NS2/NEP-C-PB1,HA-C-PB1-C-NS2/NEP, NS2/NEP-C-PB1-C-HA, PB1-C-NS2/NEP-C-HA,HA-K-NS2/NEP-C-PB1, HA-K-PB1-C-NS2/NEP, NS2/NEP-C-PB1-K-HA,PB1-C-NS2/NEP-K-HA, HA-C-NS2/NEP-K-PB1, HA-C-PB1-K-NS2/NEP,NS2/NEP-K-PB1-C-HA, PB1-K-NS2/NEP-C-HA, HA-K-NS2/NEP-PB2,HA-K-PB2-NS2/NEP, NS2/NEP-PB2-K-HA, PB2-NS2/NEP-K-HA, HA-NS2/NEP-K-PB2,HA-PB2-K-NS2/NEP, NS2/NEP-K-PB2-HA, PB2-K-NS2/NEP-HA,HA-K-NS2/NEP-K-PB2, HA-K-PB2-K-NS2/NEP, NS2/NEP-K-PB2-K-HA,PB2-K-NS2/NEP-K-HA, HA-C-NS2/NEP-PB2, HA-C-PB2-NS2/NEP,NS2/NEP-PB2-C-HA, PB2-NS2/NEP-C-HA, HA-NS2/NEP-C-PB2, HA-PB2-C-NS2/NEP,NS2/NEP-C-PB2-HA, PB2-C-NS2/NEP-HA, HA-C-NS2/NEP-C-PB2,HA-C-PB2-C-NS2/NEP, NS2/NEP-C-PB2-C-HA, PB2-C-NS2/NEP-C-HA,HA-K-NS2/NEP-C-PB2, HA-K-PB2-C-NS2/NEP, NS2/NEP-C-PB2-K-HA,PB2-C-NS2/NEP-K-HA, HA-C-NS2/NEP-K-PB2, HA-C-PB2-K-NS2/NEP,NS2/NEP-K-PB2-C-HA, PB2-K-NS2/NEP-C-HA, HA-K-NS2/NEP-PB1F2,HA-K-PB1F2-NS2/NEP, NS2/NEP-PB1F2-K-HA, PB1F2-NS2/NEP-K-HA,HA-NS2/NEP-K-PB1F2, HA-PB1F2-K-NS2/NEP, NS2/NEP-K-PB1F2-HA,PB1F2-K-NS2/NEP-HA, HA-K-NS2/NEP-K-PB1F2, HA-K-PB1F2-K-NS2/NEP,NS2/NEP-K-PB1F2-K-HA, PB1F2-K-NS2/NEP-K-HA, HA-C-NS2/NEP-PB1F2,HA-C-PB1F2-NS2/NEP, NS2/NEP-PB1F2-C-HA, PB1F2-NS2/NEP-C-HA,HA-NS2/NEP-C-PB1F2, HA-PB1F2-C-NS2/NEP, NS2/NEP-C-PB1F2-HA,PB1F2-C-NS2/NEP-HA, HA-C-NS2/NEP-C-PB1F2, HA-C-PB1F2-C-NS2/NEP,NS2/NEP-C-PB1F2-C-HA, PB1F2-C-NS2/NEP-C-HA, HA-K-NS2/NEP-C-PB1F2,HA-K-PB1F2-C-NS2/NEP, NS2/NEP-C-PB1F2-K-HA, PB1F2-C-NS2/NEP-K-HA,HA-C-NS2/NEP-K-PB1F2, HA-C-PB1F2-K-NS2/NEP, NS2/NEP-K-PB1F2-C-HA,PB1F2-K-NS2/NEP-C-HA, HA-K-PA, PA-K-HA, HA-K-PA-PA, PA-PA-K-HA,HA-PA-K-PA, PA-K-PA-HA, HA-K-PA-K-PA, PA-K-PA-K-HA, HA-C-PA, PA-C-HA,HA-C-PA-PA, PA-PA-C-HA, HA-PA-C-PA, PA-C-PA-HA, HA-C-PA-C-PA,PA-C-PA-C-HA, HA-K-PA-C-PA, PA-C-PA-K-HA, HA-C-PA-K-PA, PA-K-PA-C-HA,HA-K-PA-PB1, HA-K-PB1-PA, PA-PB1-K-HA, PB1-PA-K-HA, HA-PA-K-PB1,HA-PB1-K-PA, PA-K-PB1-HA, PB1-K-PA-HA, HA-K-PA-K-PB1, HA-K-PB1-K-PA,PA-K-PB1-K-HA, PB1-K-PA-K-HA, HA-C-PA-PB1, HA-C-PB1-PA, PA-PB1-C-HA,PB1-PA-C-HA, HA-PA-C-PB1, HA-PB1-C-PA, PA-C-PB1-HA, PB1-C-PA-HA,HA-C-PA-C-PB1, HA-C-PB1-C-PA, PA-C-PB1-C-HA, PB1-C-PA-C-HA,HA-K-PA-C-PB1, HA-K-PB1-C-PA, PA-C-PB1-K-HA, PB1-C-PA-K-HA,HA-C-PA-K-PB1, HA-C-PB1-K-PA, PA-K-PB1-C-HA, PB1-K-PA-C-HA, HA-K-PA-PB2,HA-K-PB2-PA, PA-PB2-K-HA, PB2-PA-K-HA, HA-PA-K-PB2, HA-PB2-K-PA,PA-K-PB2-HA, PB2-K-PA-HA, HA-K-PA-K-PB2, HA-K-PB2-K-PA, PA-K-PB2-K-HA,PB2-K-PA-K-HA, HA-C-PA-PB2, HA-C-PB2-PA, PA-PB2-C-HA, PB2-PA-C-HA,HA-PA-C-PB2, HA-PB2-C-PA, PA-C-PB2-HA, PB2-C-PA-HA, HA-C-PA-C-PB2,HA-C-PB2-C-PA, PA-C-PB2-C-HA, PB2-C-PA-C-HA, HA-K-PA-C-PB2,HA-K-PB2-C-PA, PA-C-PB2-K-HA, PB2-C-PA-K-HA, HA-C-PA-K-PB2,HA-C-PB2-K-PA, PA-K-PB2-C-HA, PB2-K-PA-C-HA, HA-K-PA-PB1F2,HA-K-PB1F2-PA, PA-PB1F2-K-HA, PB1F2-PA-K-HA, HA-PA-K-PB1F2,HA-PB1F2-K-PA, PA-K-PB1F2-HA, PB1F2-K-PA-HA, HA-K-PA-K-PB1F2,HA-K-PB1F2-K-PA, PA-K-PB1F2-K-HA, PB1F2-K-PA-K-HA, HA-C-PA-PB 1F2,HA-C-PB1F2-PA, PA-PB1F2-C-HA, PB1F2-PA-C-HA, HA-PA-C-PB1F2,HA-PB1F2-C-PA, PA-C-PB1F2-HA, PB1F2-C-PA-HA, HA-C-PA-C-PB1F2,HA-C-PB1F2-C-PA, PA-C-PB1F2-C-HA, PB1F2-C-PA-C-HA, HA-K-PA-C-PB1F2,HA-K-PB1F2-C-PA, PA-C-PB1F2-K-HA, PB1F2-C-PA-K-HA, HA-C-PA-K-PB1F2,HA-C-PB1F2-K-PA, PA-K-PB1F2-C-HA, PB1F2-K-PA-C-HA, HA-K-PB1, PB1-K-HA,HA-K-PB1-PB1, PB1-PB1-K-HA, HA-PB1-K-PB1, PB1-K-PB1-HA, HA-K-PB1-K-PB1,PB1-K-PB1-K-HA, HA-C-PB1, PB1-C-HA, HA-C-PB1-PB1, PB1-PB1-C-HA,HA-PB1-C-PB1, PB1-C-PB1-HA, HA-C-PB1-C-PB1, PB1-C-PB1-C-HA,HA-K-PB1-C-PB1, PB1-C-PB1-K-HA, HA-C-PB1-K-PB1, PB1-K-PB1-C-HA,HA-K-PB1-PB2, HA-K-PB2-PB1, PB1-PB2-K-HA, PB2-PB1-K-HA, HA-PB1-K-PB2,HA-PB2-K-PB1, PB1-K-PB2-HA, PB2-K-PB1-HA, HA-K-PB1-K-PB2,HA-K-PB2-K-PB1, PB1-K-PB2-K-HA, PB2-K-PB1-K-HA, HA-C-PB1-PB2,HA-C-PB2-PB1, PB1-PB2-C-HA, PB2-PB1-C-HA, HA-PB1-C-PB2, HA-PB2-C-PB1,PB1-C-PB2-HA, PB2-C-PB1-HA, HA-C-PB1-C-PB2, HA-C-PB2-C-PB1,PB1-C-PB2-C-HA, PB2-C-PB1-C-HA, HA-K-PB1-C-PB2, HA-K-PB2-C-PB1,PB1-C-PB2-K-HA, PB2-C-PB1-K-HA, HA-C-PB1-K-PB2, HA-C-PB2-K-PB1,PB1-K-PB2-C-HA, PB2-K-PB1-C-HA, HA-K-PB1-PB1F2, HA-K-PB1F2-PB1,PB1-PB1F2-K-HA, PB1F2-PB1-K-HA, HA-PB1-K-PB1F2, HA-PB1F2-K-PB1,PB1-K-PB1F2-HA, PB1F2-K-PB1-HA, HA-K-PB1-K-PB1F2, HA-K-PB1F2-K-PB1,PB1-K-PB1F2-K-HA, PB1F2-K-PB1-K-HA, HA-C-PB1-PB1F2, HA-C-PB1F2-PB1,PB1-PB1F2-C-HA, PB1F2-PB1-C-HA, HA-PB1-C-PB1F2, HA-PB1F2-C-PB1,PB1-C-PB1F2-HA, PB1F2-C-PB1-HA, HA-C-PB1-C-PB1F2, HA-C-PB1F2-C-PB1,PB1-C-PB1F2-C-HA, PB1F2-C-PB1-C-HA, HA-K-PB1-C-PB1F2, HA-K-PB 1F2-C-PB1,PB1-C-PB1F2-K-HA, PB1F2-C-PB1-K-HA, HA-C-PB1-K-PB1F2, HA-C-PB1F2-K-PB1,PB1-K-PB1F2-C-HA, PB1F2-K-PB1-C-HA, HA-K-PB2, PB2-K-HA, HA-K-PB2-PB2,PB2-PB2-K-HA, HA-PB2-K-PB2, PB2-K-PB2-HA, HA-K-PB2-K-PB2,PB2-K-PB2-K-HA, HA-C-PB2, PB2-C-HA, HA-C-PB2-PB2, PB2-PB2-C-HA,HA-PB2-C-PB2, PB2-C-PB2-HA, HA-C-PB2-C-PB2, PB2-C-PB2-C-HA,HA-K-PB2-C-PB2, PB2-C-PB2-K-HA, HA-C-PB2-K-PB2, PB2-K-PB2-C-HA,HA-K-PB2-PB1F2, HA-K-PB1F2-PB2, PB2-PB1F2-K-HA, PB1F2-PB2-K-HA,HA-PB2-K-PB1F2, HA-PB1F2-K-PB2, PB2-K-PB1F2-HA, PB1F2-K-PB2-HA,HA-K-PB2-K-PB1F2, HA-K-PB1F2-K-PB2, PB2-K-PB1F2-K-HA, PB1F2-K-PB2-K-HA,HA-C-PB2-PB1F2, HA-C-PB1F2-PB2, PB2-PB1F2-C-HA, PB1F2-PB2-C-HA,HA-PB2-C-PB1F2, HA-PB1F2-C-PB2, PB2-C-PB1F2-HA, PB1F2-C-PB2-HA,HA-C-PB2-C-PB1F2, HA-C-PB 1F2-C-PB2, PB2-C-PB1F2-C-HA, PB1F2-C-PB2-C-HA,HA-K-PB2-C-PB1F2, HA-K-PB1F2-C-PB2, PB2-C-PB1F2-K-HA, PB1F2-C-PB2-K-HA,HA-C-PB2-K-PB1F2, HA-C-PB1F2-K-PB2, PB2-K-PB1F2-C-HA, PB1F2-K-PB2-C-HA,HA-K-PB1F2, PB1F2-K-HA, HA-K-PB 1F2-PB1F2, PB1F2-PB1F2-K-HA,HA-PB1F2-K-PB1F2, PB1F2-K-PB1F2-HA, HA-K-PB1F2-K-PB1F2,PB1F2-K-PB1F2-K-HA, HA-C-PB1F2, PB1F2-C-HA, HA-C-PB1F2-PB1F2,PB1F2-PB1F2-C-HA, HA-PB1F2-C-PB1F2, PB1F2-C-PB1F2-HA,HA-C-PB1F2-C-PB1F2, PB1F2-C-PB1F2-C-HA, HA-K-PB1F2-C-PB1F2,PB1F2-C-PB1F2-K-HA, HA-C-PB1F2-K-PB1F2, PB1F2-K-PB1F2-C-HA, NA-K-NP,NP-K-NA, NA-K-NP-NP, NP-NP-K-NA, NA-NP-K-NP, NP-K-NP-NA, NA-K-NP-K-NP,NP-K-NP-K-NA, NA-C-NP, NP-C-NA, NA-C-NP-NP, NP-NP-C-NA, NA-NP-C-NP,NP-C-NP-NA, NA-C-NP-C-NP, NP-C-NP-C-NA, NA-K-NP-C-NP, NP-C-NP-K-NA,NA-C-NP-K-NP, NP-K-NP-C-NA, NA-K-NP-M1, NA-K-M1-NP, NP-M1-K-NA,M1-NP-K-NA, NA-NP-K-M1, NA-M1-K-NP, NP-K-M1-NA, M1-K-NP-NA,NA-K-NP-K-M1, NA-K-M1-K-NP, NP-K-M1-K-NA, M1-K-NP-K-NA, NA-C-NP-M1,NA-C-M1-NP, NP-M1-C-NA, M1-NP-C-NA, NA-NP-C-M1, NA-M1-C-NP, NP-C-M1-NA,M1-C-NP-NA, NA-C-NP-C-M1, NA-C-M1-C-NP, NP-C-M1-C-NA, M1-C-NP-C-NA,NA-K-NP-C-M1, NA-K-M1-C-NP, NP-C-M1-K-NA, M1-C-NP-K-NA, NA-C-NP-K-M1,NA-C-M1-K-NP, NP-K-M1-C-NA, M1-K-NP-C-NA, NA-K-NP-M2, NA-K-M2-NP,NP-M2-K-NA, M2-NP-K-NA, NA-NP-K-M2, NA-M2-K-NP, NP-K-M2-NA, M2-K-NP-NA,NA-K-NP-K-M2, NA-K-M2-K-NP, NP-K-M2-K-NA, M2-K-NP-K-NA, NA-C-NP-M2,NA-C-M2-NP, NP-M2-C-NA, M2-NP-C-NA, NA-NP-C-M2, NA-M2-C-NP, NP-C-M2-NA,M2-C-NP-NA, NA-C-NP-C-M2, NA-C-M2-C-NP, NP-C-M2-C-NA, M2-C-NP-C-NA,NA-K-NP-C-M2, NA-K-M2-C-NP, NP-C-M2-K-NA, M2-C-NP-K-NA, NA-C-NP-K-M2,NA-C-M2-K-NP, NP-K-M2-C-NA, M2-K-NP-C-NA, NA-K-NP-NS1, NA-K-NS1-NP,NP-NS1-K-NA, NS1-NP-K-NA, NA-NP-K-NS1, NA-NS1-K-NP, NP-K-NS1-NA,NS1-K-NP-NA, NA-K-NP-K-NS1, NA-K-NS1-K-NP, NP-K-NS1-K-NA, NS1-K-NP-K-NA,NA-C-NP-NS1, NA-C-NS1-NP, NP-NS1-C-NA, NS1-NP-C-NA, NA-NP-C-NS1,NA-NS1-C-NP, NP-C-NS1-NA, NS1-C-NP-NA, NA-C-NP-C-NS1, NA-C-NS1-C-NP,NP-C-NS1-C-NA, NS1-C-NP-C-NA, NA-K-NP-C-NS1, NA-K-NS1-C-NP,NP-C-NS1-K-NA, NS1-C-NP-K-NA, NA-C-NP-K-NS1, NA-C-NS1-K-NP,NP-K-NS1-C-NA, NS1-K-NP-C-NA, NA-K-NP-NS2/NEP, NA-K-NS2/NEP-NP,NP-NS2/NEP-K-NA, NS2/NEP-NP-K-NA, NA-NP-K-NS2/NEP, NA-NS2/NEP-K-NP,NP-K-NS2/NEP-NA, NS2/NEP-K-NP-NA, NA-K-NP-K-NS2/NEP, NA-K-NS2/NEP-K-NP,NP-K-NS2/NEP-K-NA, NS2/NEP-K-NP-K-NA, NA-C-NP-NS2/NEP, NA-C-NS2/NEP-NP,NP-NS2/NEP-C-NA, NS2/NEP-NP-C-NA, NA-NP-C-NS2/NEP, NA-NS2/NEP-C-NP,NP-C-NS2/NEP-NA, NS2/NEP-C-NP-NA, NA-C-NP-C-NS2/NEP, NA-C-NS2/NEP-C-NP,NP-C-NS2/NEP-C-NA, NS2/NEP-C-NP-C-NA, NA-K-NP-C-NS2/NEP,NA-K-NS2/NEP-C-NP, NP-C-NS2/NEP-K-NA, NS2/NEP-C-NP-K-NA,NA-C-NP-K-NS2/NEP, NA-C-NS2/NEP-K-NP, NP-K-NS2/NEP-C-NA,NS2/NEP-K-NP-C-NA, NA-K-NP-PA, NA-K-PA-NP, NP-PA-K-NA, PA-NP-K-NA,NA-NP-K-PA, NA-PA-K-NP, NP-K-PA-NA, PA-K-NP-NA, NA-K-NP-K-PA,NA-K-PA-K-NP, NP-K-PA-K-NA, PA-K-NP-K-NA, NA-C-NP-PA, NA-C-PA-NP,NP-PA-C-NA, PA-NP-C-NA, NA-NP-C-PA, NA-PA-C-NP, NP-C-PA-NA, PA-C-NP-NA,NA-C-NP-C-PA, NA-C-PA-C-NP, NP-C-PA-C-NA, PA-C-NP-C-NA, NA-K-NP-C-PA,NA-K-PA-C-NP, NP-C-PA-K-NA, PA-C-NP-K-NA, NA-C-NP-K-PA, NA-C-PA-K-NP,NP-K-PA-C-NA, PA-K-NP-C-NA, NA-K-NP-PB1, NA-K-PB1-NP, NP-PB1-K-NA,PB1-NP-K-NA, NA-NP-K-PB1, NA-PB1-K-NP, NP-K-PB1-NA, PB1-K-NP-NA,NA-K-NP-K-PB1, NA-K-PB1-K-NP, NP-K-PB1-K-NA, PB1-K-NP-K-NA, NA-C-NP-PB1,NA-C-PB1-NP, NP-PB1-C-NA, PB1-NP-C-NA, NA-NP-C-PB1, NA-PB1-C-NP,NP-C-PB1-NA, PB1-C-NP-NA, NA-C-NP-C-PB1, NA-C-PB1-C-NP, NP-C-PB1-C-NA,PB1-C-NP-C-NA, NA-K-NP-C-PB1, NA-K-PB1-C-NP, NP-C-PB1-K-NA,PB1-C-NP-K-NA, NA-C-NP-K-PB1, NA-C-PB1-K-NP, NP-K-PB1-C-NA,PB1-K-NP-C-NA, NA-K-NP-PB2, NA-K-PB2-NP, NP-PB2-K-NA, PB2-NP-K-NA,NA-NP-K-PB2, NA-PB2-K-NP, NP-K-PB2-NA, PB2-K-NP-NA, NA-K-NP-K-PB2,NA-K-PB2-K-NP, NP-K-PB2-K-NA, PB2-K-NP-K-NA, NA-C-NP-PB2, NA-C-PB2-NP,NP-PB2-C-NA, PB2-NP-C-NA, NA-NP-C-PB2, NA-PB2-C-NP, NP-C-PB2-NA,PB2-C-NP-NA, NA-C-NP-C-PB2, NA-C-PB2-C-NP, NP-C-PB2-C-NA, PB2-C-NP-C-NA,NA-K-NP-C-PB2, NA-K-PB2-C-NP, NP-C-PB2-K-NA, PB2-C-NP-K-NA,NA-C-NP-K-PB2, NA-C-PB2-K-NP, NP-K-PB2-C-NA, PB2-K-NP-C-NA,NA-K-NP-PB1F2, NA-K-PB1F2-NP, NP-PB1F2-K-NA, PB1F2-NP-K-NA,NA-NP-K-PB1F2, NA-PB1F2-K-NP, NP-K-PB1F2-NA, PB1F2-K-NP-NA,NA-K-NP-K-PB1F2, NA-K-PB1F2-K-NP, NP-K-PB1F2-K-NA, PB1F2-K-NP-K-NA,NA-C-NP-PB1F2, NA-C-PB1F2-NP, NP-PB1F2-C-NA, PB1F2-NP-C-NA,NA-NP-C-PB1F2, NA-PB1F2-C-NP, NP-C-PB1F2-NA, PB1F2-C-NP-NA,NA-C-NP-C-PB1F2, NA-C-PB1F2-C-NP, NP-C-PB1F2-C-NA, PB1F2-C-NP-C-NA,NA-K-NP-C-PB1F2, NA-K-PB1F2-C-NP, NP-C-PB1F2-K-NA, PB1F2-C-NP-K-NA,NA-C-NP-K-PB1F2, NA-C-PB1F2-K-NP, NP-K-PB1F2-C-NA, PB1F2-K-NP-C-NA,NA-K-M1, M1-K-NA, NA-K-M1-M1, M1-M1-K-NA, NA-M1-K-M1, M1-K-M1-NA,NA-K-M1-K-M1, M1-K-M1-K-NA, NA-C-M1, M1-C-NA, NA-C-M1-M1, M1-M1-C-NA,NA-M1-C-M1, M1-C-M1-NA, NA-C-M1-C-M1, M1-C-M1-C-NA, NA-K-M1-C-M1,M1-C-M1-K-NA, NA-C-M1-K-M1, M1-K-M1-C-NA, NA-K-M1-M2, NA-K-M2-M1,M1-M2-K-NA, M2-M1-K-NA, NA-M1-K-M2, NA-M2-K-M1, M1-K-M2-NA, M2-K-M1-NA,NA-K-M1-K-M2, NA-K-M2-K-M1, M1-K-M2-K-NA, M2-K-M1-K-NA, NA-C-M1-M2,NA-C-M2-M1, M1-M2-C-NA, M2-M1-C-NA, NA-M1-C-M2, NA-M2-C-M1, M1-C-M2-NA,M2-C-M1-NA, NA-C-M1-C-M2, NA-C-M2-C-M1, M1-C-M2-C-NA, M2-C-M1-C-NA,NA-K-M1-C-M2, NA-K-M2-C-M1, M1-C-M2-K-NA, M2-C-M1-K-NA, NA-C-M1-K-M2,NA-C-M2-K-M1, M1-K-M2-C-NA, M2-K-M1-C-NA, NA-K-M1-NS1, NA-K-NS1-M1,M1-NS1-K-NA, NS1-M1-K-NA, NA-M1-K-NS1, NA-NS1-K-M1, M1-K-NS1-NA,NS1-K-M1-NA, NA-K-M1-K-NS1, NA-K-NS1-K-M1, M1-K-NS1-K-NA, NS1-K-M1-K-NA,NA-C-M1-NS1, NA-C-NS1-M1, M1-NS1-C-NA, NS1-M1-C-NA, NA-M1-C-NS1,NA-NS1-C-M1, M1-C-NS1-NA, NS1-C-M1-NA, NA-C-M1-C-NS1, NA-C-NS1-C-M1,M1-C-NS1-C-NA, NS1-C-M1-C-NA, NA-K-M1-C-NS1, NA-K-NS1-C-M1,M1-C-NS1-K-NA, NS1-C-M1-K-NA, NA-C-M1-K-NS1, NA-C-NS1-K-M1,M1-K-NS1-C-NA, NS1-K-M1-C-NA, NA-K-M1-NS2/NEP, NA-K-NS2/NEP-M1,M1-NS2/NEP-K-NA, NS2/NEP-M1-K-NA, NA-M1-K-NS2/NEP, NA-NS2/NEP-K-M1,M1-K-NS2/NEP-NA, NS2/NEP-K-M1-NA, NA-K-M1-K-NS2/NEP, NA-K-NS2/NEP-K-M1,M1-K-NS2/NEP-K-NA, NS2/NEP-K-M1-K-NA, NA-C-M1-NS2/NEP, NA-C-NS2/NEP-M1,M1-NS2/NEP-C-NA, NS2/NEP-M1-C-NA, NA-M1-C-NS2/NEP, NA-NS2/NEP-C-M1,M1-C-NS2/NEP-NA, NS2/NEP-C-M1-NA, NA-C-M1-C-NS2/NEP, NA-C-NS2/NEP-C-M1,M1-C-NS2/NEP-C-NA, NS2/NEP-C-M1-C-NA, NA-K-M1-C-NS2/NEP,NA-K-NS2/NEP-C-M1, M1-C-NS2/NEP-K-NA, NS2/NEP-C-M1-K-NA,NA-C-M1-K-NS2/NEP, NA-C-NS2/NEP-K-M1, M1-K-NS2/NEP-C-NA,NS2/NEP-K-M1-C-NA, NA-K-M1-PA, NA-K-PA-M1, M1-PA-K-NA, PA-M1-K-NA,NA-M1-K-PA, NA-PA-K-M1, M1-K-PA-NA, PA-K-M1-NA, NA-K-M1-K-PA,NA-K-PA-K-M1, M1-K-PA-K-NA, PA-K-M1-K-NA, NA-C-M1-PA, NA-C-PA-M1,M1-PA-C-NA, PA-M1-C-NA, NA-M1-C-PA, NA-PA-C-M1, M1-C-PA-NA, PA-C-M1-NA,NA-C-M1-C-PA, NA-C-PA-C-M1, M1-C-PA-C-NA, PA-C-M1-C-NA, NA-K-M1-C-PA,NA-K-PA-C-M1, M1-C-PA-K-NA, PA-C-M1-K-NA, NA-C-M1-K-PA, NA-C-PA-K-M1,M1-K-PA-C-NA, PA-K-M1-C-NA, NA-K-M1-PB1, NA-K-PB1-M1, M1-PB1-K-NA,PB1-M1-K-NA, NA-M1-K-PB1, NA-PB1-K-M1, M1-K-PB1-NA, PB1-K-M1-NA,NA-K-M1-K-PB1, NA-K-PB1-K-M1, M1-K-PB1-K-NA, PB1-K-M1-K-NA, NA-C-M1-PB1,NA-C-PB1-M1, M1-PB1-C-NA, PB1-M1-C-NA, NA-M1-C-PB1, NA-PB1-C-M1,M1-C-PB1-NA, PB1-C-M1-NA, NA-C-M1-C-PB1, NA-C-PB1-C-M1, M1-C-PB1-C-NA,PB1-C-M1-C-NA, NA-K-M1-C-PB1, NA-K-PB1-C-M1, M1-C-PB1-K-NA,PB1-C-M1-K-NA, NA-C-M1-K-PB1, NA-C-PB1-K-M1, M1-K-PB1-C-NA,PB1-K-M1-C-NA, NA-K-M1-PB2, NA-K-PB2-M1, M1-PB2-K-NA, PB2-M1-K-NA,NA-M1-K-PB2, NA-PB2-K-M1, M1-K-PB2-NA, PB2-K-M1-NA, NA-K-M1-K-PB2,NA-K-PB2-K-M1, M1-K-PB2-K-NA, PB2-K-M1-K-NA, NA-C-M1-PB2, NA-C-PB2-M1,M1-PB2-C-NA, PB2-M1-C-NA, NA-M1-C-PB2, NA-PB2-C-M1, M1-C-PB2-NA,PB2-C-M1-NA, NA-C-M1-C-PB2, NA-C-PB2-C-M1, M1-C-PB2-C-NA, PB2-C-M1-C-NA,NA-K-M1-C-PB2, NA-K-PB2-C-M1, M1-C-PB2-K-NA, PB2-C-M1-K-NA,NA-C-M1-K-PB2, NA-C-PB2-K-M1, M1-K-PB2-C-NA, PB2-K-M1-C-NA,NA-K-M1-PB1F2, NA-K-PB1F2-M1, M1-PB1F2-K-NA, PB1F2-M1-K-NA,NA-M1-K-PB1F2, NA-PB1F2-K-M1, M1-K-PB1F2-NA, PB1F2-K-M1-NA,NA-K-M1-K-PB1F2, NA-K-PB1F2-K-M1, M1-K-PB1F2-K-NA, PB1F2-K-M1-K-NA,NA-C-M1-PB1F2, NA-C-PB1F2-M1, M1-PB1F2-C-NA, PB1F2-M1-C-NA,NA-M1-C-PB1F2, NA-PB1F2-C-M1, M1-C-PB1F2-NA, PB1F2-C-M1-NA,NA-C-M1-C-PB1F2, NA-C-PB1F2-C-M1, M1-C-PB1F2-C-NA, PB1F2-C-M1-C-NA,NA-K-M1-C-PB1F2, NA-K-PB1F2-C-M1, M1-C-PB1F2-K-NA, PB1F2-C-M1-K-NA,NA-C-M1-K-PB1F2, NA-C-PB1F2-K-M1, M1-K-PB1F2-C-NA, PB1F2-K-M1-C-NA,NA-K-M2, M2-K-NA, NA-K-M2-M2, M2-M2-K-NA, NA-M2-K-M2, M2-K-M2-NA,NA-K-M2-K-M2, M2-K-M2-K-NA, NA-C-M2, M2-C-NA, NA-C-M2-M2, M2-M2-C-NA,NA-M2-C-M2, M2-C-M2-NA, NA-C-M2-C-M2, M2-C-M2-C-NA, NA-K-M2-C-M2,M2-C-M2-K-NA, NA-C-M2-K-M2, M2-K-M2-C-NA, NA-K-M2-NS1, NA-K-NS1-M2,M2-NS1-K-NA, NS1-M2-K-NA, NA-M2-K-NS1, NA-NS1-K-M2, M2-K-NS1-NA,NS1-K-M2-NA, NA-K-M2-K-NS1, NA-K-NS1-K-M2, M2-K-NS1-K-NA, NS1-K-M2-K-NA,NA-C-M2-NS1, NA-C-NS1-M2, M2-NS1-C-NA, NS1-M2-C-NA, NA-M2-C-NS1,NA-NS1-C-M2, M2-C-NS1-NA, NS1-C-M2-NA, NA-C-M2-C-NS1, NA-C-NS1-C-M2,M2-C-NS1-C-NA, NS1-C-M2-C-NA, NA-K-M2-C-NS1, NA-K-NS1-C-M2,M2-C-NS1-K-NA, NS1-C-M2-K-NA, NA-C-M2-K-NS1, NA-C-NS1-K-M2,M2-K-NS1-C-NA, NS1-K-M2-C-NA, NA-K-M2-NS2/NEP, NA-K-NS2/NEP-M2,M2-NS2/NEP-K-NA, NS2/NEP-M2-K-NA, NA-M2-K-NS2/NEP, NA-NS2/NEP-K-M2,M2-K-NS2/NEP-NA, NS2/NEP-K-M2-NA, NA-K-M2-K-NS2/NEP, NA-K-NS2/NEP-K-M2,M2-K-NS2/NEP-K-NA, NS2/NEP-K-M2-K-NA, NA-C-M2-NS2/NEP, NA-C-NS2/NEP-M2,M2-NS2/NEP-C-NA, NS2/NEP-M2-C-NA, NA-M2-C-NS2/NEP, NA-NS2/NEP-C-M2,M2-C-NS2/NEP-NA, NS2/NEP-C-M2-NA, NA-C-M2-C-NS2/NEP, NA-C-NS2/NEP-C-M2,M2-C-NS2/NEP-C-NA, NS2/NEP-C-M2-C-NA, NA-K-M2-C-NS2/NEP,NA-K-NS2/NEP-C-M2, M2-C-NS2/NEP-K-NA, NS2/NEP-C-M2-K-NA,NA-C-M2-K-NS2/NEP, NA-C-NS2/NEP-K-M2, M2-K-NS2/NEP-C-NA,NS2/NEP-K-M2-C-NA, NA-K-M2-PA, NA-K-PA-M2, M2-PA-K-NA, PA-M2-K-NA,NA-M2-K-PA, NA-PA-K-M2, M2-K-PA-NA, PA-K-M2-NA, NA-K-M2-K-PA,NA-K-PA-K-M2, M2-K-PA-K-NA, PA-K-M2-K-NA, NA-C-M2-PA, NA-C-PA-M2,M2-PA-C-NA, PA-M2-C-NA, NA-M2-C-PA, NA-PA-C-M2, M2-C-PA-NA, PA-C-M2-NA,NA-C-M2-C-PA, NA-C-PA-C-M2, M2-C-PA-C-NA, PA-C-M2-C-NA, NA-K-M2-C-PA,NA-K-PA-C-M2, M2-C-PA-K-NA, PA-C-M2-K-NA, NA-C-M2-K-PA, NA-C-PA-K-M2,M2-K-PA-C-NA, PA-K-M2-C-NA, NA-K-M2-PB1, NA-K-PB1-M2, M2-PB1-K-NA,PB1-M2-K-NA, NA-M2-K-PB1, NA-PB1-K-M2, M2-K-PB1-NA, PB1-K-M2-NA,NA-K-M2-K-PB1, NA-K-PB1-K-M2, M2-K-PB1-K-NA, PB1-K-M2-K-NA, NA-C-M2-PB1,NA-C-PB1-M2, M2-PB1-C-NA, PB1-M2-C-NA, NA-M2-C-PB1, NA-PB1-C-M2,M2-C-PB1-NA, PB1-C-M2-NA, NA-C-M2-C-PB1, NA-C-PB1-C-M2, M2-C-PB1-C-NA,PB1-C-M2-C-NA, NA-K-M2-C-PB1, NA-K-PB1-C-M2, M2-C-PB1-K-NA,PB1-C-M2-K-NA, NA-C-M2-K-PB1, NA-C-PB1-K-M2, M2-K-PB1-C-NA,PB1-K-M2-C-NA, NA-K-M2-PB2, NA-K-PB2-M2, M2-PB2-K-NA, PB2-M2-K-NA,NA-M2-K-PB2, NA-PB2-K-M2, M2-K-PB2-NA, PB2-K-M2-NA, NA-K-M2-K-PB2,NA-K-PB2-K-M2, M2-K-PB2-K-NA, PB2-K-M2-K-NA, NA-C-M2-PB2, NA-C-PB2-M2,M2-PB2-C-NA, PB2-M2-C-NA, NA-M2-C-PB2, NA-PB2-C-M2, M2-C-PB2-NA,PB2-C-M2-NA, NA-C-M2-C-PB2, NA-C-PB2-C-M2, M2-C-PB2-C-NA, PB2-C-M2-C-NA,NA-K-M2-C-PB2, NA-K-PB2-C-M2, M2-C-PB2-K-NA, PB2-C-M2-K-NA,NA-C-M2-K-PB2, NA-C-PB2-K-M2, M2-K-PB2-C-NA, PB2-K-M2-C-NA,NA-K-M2-PB1F2, NA-K-PB1F2-M2, M2-PB1F2-K-NA, PB1F2-M2-K-NA,NA-M2-K-PB1F2, NA-PB1F2-K-M2, M2-K-PB1F2-NA, PB1F2-K-M2-NA,NA-K-M2-K-PB1F2, NA-K-PB1F2-K-M2, M2-K-PB1F2-K-NA, PB1F2-K-M2-K-NA,NA-C-M2-PB1F2, NA-C-PB1F2-M2, M2-PB1F2-C-NA, PB1F2-M2-C-NA,NA-M2-C-PB1F2, NA-PB1F2-C-M2, M2-C-PB1F2-NA, PB1F2-C-M2-NA,NA-C-M2-C-PB1F2, NA-C-PB1F2-C-M2, M2-C-PB1F2-C-NA, PB1F2-C-M2-C-NA,NA-K-M2-C-PB1F2, NA-K-PB1F2-C-M2, M2-C-PB1F2-K-NA, PB1F2-C-M2-K-NA,NA-C-M2-K-PB1F2, NA-C-PB1F2-K-M2, M2-K-PB1F2-C-NA, PB1F2-K-M2-C-NA,NA-K-NS1, NS1-K-NA, NA-K-NS1-NS1, NS1-NS1-K-NA, NA-NS1-K-NS1,NS1-K-NS1-NA, NA-K-NS1-K-NS1, NS1-K-NS1-K-NA, NA-C-NS1, NS1-C-NA,NA-C-NS1-NS1, NS1-NS1-C-NA, NA-NS1-C-NS1, NS1-C-NS1-NA, NA-C-NS1-C-NS1,NS1-C-NS1-C-NA, NA-K-NS1-C-NS1, NS1-C-NS1-K-NA, NA-C-NS1-K-NS1,NS1-K-NS1-C-NA, NA-K-NS1-NS2/NEP, NA-K-NS2/NEP-NS1, NS1-NS2/NEP-K-NA,NS2/NEP-NS1-K-NA, NA-NS1-K-NS2/NEP, NA-NS2/NEP-K-NS1, NS1-K-NS2/NEP-NA,NS2/NEP-K-NS1-NA, NA-K-NS1-K-NS2/NEP, NA-K-NS2/NEP-K-NS1,NS1-K-NS2/NEP-K-NA, NS2/NEP-K-NS1-K-NA, NA-C-NS1-NS2/NEP,NA-C-NS2/NEP-NS1, NS1-NS2/NEP-C-NA, NS2/NEP-NS1-C-NA, NA-NS1-C-NS2/NEP,NA-NS2/NEP-C-NS1, NS1-C-NS2/NEP-NA, NS2/NEP-C-NS1-NA,NA-C-NS1-C-NS2/NEP, NA-C-NS2/NEP-C-NS1, NS1-C-NS2/NEP-C-NA,NS2/NEP-C-NS1-C-NA, NA-K-NS1-C-NS2/NEP, NA-K-NS2/NEP-C-NS1,NS1-C-NS2/NEP-K-NA, NS2/NEP-C-NS1-K-NA, NA-C-NS1-K-NS2/NEP,NA-C-NS2/NEP-K-NS1, NS1-K-NS2/NEP-C-NA, NS2/NEP-K-NS1-C-NA, NA-K-NS1-PA,NA-K-PA-NS1, NS1-PA-K-NA, PA-NS1-K-NA, NA-NS1-K-PA, NA-PA-K-NS1,NS1-K-PA-NA, PA-K-NS1-NA, NA-K-NS1-K-PA, NA-K-PA-K-NS1, NS1-K-PA-K-NA,PA-K-NS1-K-NA, NA-C-NS1-PA, NA-C-PA-NS1, NS1-PA-C-NA, PA-NS1-C-NA,NA-NS1-C-PA, NA-PA-C-NS1, NS1-C-PA-NA, PA-C-NS1-NA, NA-C-NS1-C-PA,NA-C-PA-C-NS1, NS1-C-PA-C-NA, PA-C-NS1-C-NA, NA-K-NS1-C-PA,NA-K-PA-C-NS1, NS1-C-PA-K-NA, PA-C-NS1-K-NA, NA-C-NS1-K-PA,NA-C-PA-K-NS1, NS1-K-PA-C-NA, PA-K-NS1-C-NA, NA-K-NS1-PB1, NA-K-PB1-NS1,NS1-PB1-K-NA, PB1-NS1-K-NA, NA-NS1-K-PB1, NA-PB1-K-NS1, NS1-K-PB1-NA,PB1-K-NS1-NA, NA-K-NS1-K-PB1, NA-K-PB1-K-NS1, NS1-K-PB1-K-NA,PB1-K-NS1-K-NA, NA-C-NS1-PB1, NA-C-PB1-NS1, NS1-PB1-C-NA, PB1-NS1-C-NA,NA-NS1-C-PB1, NA-PB1-C-NS1, NS1-C-PB1-NA, PB1-C-NS1-NA, NA-C-NS1-C-PB1,NA-C-PB1-C-NS1, NS1-C-PB1-C-NA, PB1-C-NS1-C-NA, NA-K-NS1-C-PB1,NA-K-PB1-C-NS1, NS1-C-PB1-K-NA, PB1-C-NS1-K-NA, NA-C-NS1-K-PB1,NA-C-PB1-K-NS1, NS1-K-PB1-C-NA, PB1-K-NS1-C-NA, NA-K-NS1-PB2,NA-K-PB2-NS1, NS1-PB2-K-NA, PB2-NS1-K-NA, NA-NS1-K-PB2, NA-PB2-K-NS1,NS1-K-PB2-NA, PB2-K-NS1-NA, NA-K-NS1-K-PB2, NA-K-PB2-K-NS1,NS1-K-PB2-K-NA, PB2-K-NS1-K-NA, NA-C-NS1-PB2, NA-C-PB2-NS1,NS1-PB2-C-NA, PB2-NS1-C-NA, NA-NS1-C-PB2, NA-PB2-C-NS1, NS1-C-PB2-NA,PB2-C-NS1-NA, NA-C-NS1-C-PB2, NA-C-PB2-C-NS1, NS1-C-PB2-C-NA,PB2-C-NS1-C-NA, NA-K-NS1-C-PB2, NA-K-PB2-C-NS1, NS1-C-PB2-K-NA,PB2-C-NS1-K-NA, NA-C-NS1-K-PB2, NA-C-PB2-K-NS1, NS1-K-PB2-C-NA,PB2-K-NS1-C-NA, NA-K-NS1-PB1F2, NA-K-PB1F2-NS1, NS1-PB1F2-K-NA,PB1F2-NS1-K-NA, NA-NS1-K-PB1F2, NA-PB1F2-K-NS1, NS1-K-PB1F2-NA,PB1F2-K-NS1-NA, NA-K-NS1-K-PB1F2, NA-K-PB1F2-K-NS1, NS1-K-PB1F2-K-NA,PB1F2-K-NS1-K-NA, NA-C-NS1-PB1F2, NA-C-PB1F2-NS1, NS1-PB1F2-C-NA,PB1F2-NS1-C-NA, NA-NS1-C-PB1F2, NA-PB1F2-C-NS1, NS1-C-PB1F2-NA,PB1F2-C-NS1-NA, NA-C-NS1-C-PB1F2, NA-C-PB1F2-C-NS1, NS1-C-PB1F2-C-NA,PB1F2-C-NS1-C-NA, NA-K-NS1-C-PB1F2, NA-K-PB1F2-C-NS1, NS1-C-PB1F2-K-NA,PB1F2-C-NS1-K-NA, NA-C-NS1-K-PB1F2, NA-C-PB1F2-K-NS1, NS1-K-PB1F2-C-NA,PB1F2-K-NS1-C-NA, NA-K-NS2/NEP, NS2/NEP-K-NA, NA-K-NS2/NEP-NS2/NEP,NS2/NEP-NS2/NEP-K-NA, NA-NS2/NEP-K-NS2/NEP, NS2/NEP-K-NS2/NEP-NA,NA-K-NS2/NEP-K-NS2/NEP, NS2/NEP-K-NS2/NEP-K-NA, NA-C-NS2/NEP,NS2/NEP-C-NA, NA-C-NS2/NEP-NS2/NEP, NS2/NEP-NS2/NEP-C-NA,NA-NS2/NEP-C-NS2/NEP, NS2/NEP-C-NS2/NEP-NA, NA-C-NS2/NEP-C-NS2/NEP,NS2/NEP-C-NS2/NEP-C-NA, NA-K-NS2/NEP-C-NS2/NEP, NS2/NEP-C-NS2/NEP-K-NA,NA-C-NS2/NEP-K-NS2/NEP, NS2/NEP-K-NS2/NEP-C-NA, NA-K-NS2/NEP-PA,NA-K-PA-NS2/NEP, NS2/NEP-PA-K-NA, PA-NS2/NEP-K-NA, NA-NS2/NEP-K-PA,NA-PA-K-NS2/NEP, NS2/NEP-K-PA-NA, PA-K-NS2/NEP-NA, NA-K-NS2/NEP-K-PA,NA-K-PA-K-NS2/NEP, NS2/NEP-K-PA-K-NA, PA-K-NS2/NEP-K-NA,NA-C-NS2/NEP-PA, NA-C-PA-NS2/NEP, NS2/NEP-PA-C-NA, PA-NS2/NEP-C-NA,NA-NS2/NEP-C-PA, NA-PA-C-NS2/NEP, NS2/NEP-C-PA-NA, PA-C-NS2/NEP-NA,NA-C-NS2/NEP-C-PA, NA-C-PA-C-NS2/NEP, NS2/NEP-C-PA-C-NA,PA-C-NS2/NEP-C-NA, NA-K-NS2/NEP-C-PA, NA-K-PA-C-NS2/NEP,NS2/NEP-C-PA-K-NA, PA-C-NS2/NEP-K-NA, NA-C-NS2/NEP-K-PA,NA-C-PA-K-NS2/NEP, NS2/NEP-K-PA-C-NA, PA-K-NS2/NEP-C-NA,NA-K-NS2/NEP-PB1, NA-K-PB 1-NS2/NEP, NS2/NEP-PB1-K-NA, PB1-NS2/NEP-K-NA,NA-NS2/NEP-K-PB1, NA-PB1-K-NS2/NEP, NS2/NEP-K-PB1-NA, PB1-K-NS2/NEP-NA,NA-K-NS2/NEP-K-PB1, NA-K-PB 1-K-NS2/NEP, NS2/NEP-K-PB1-K-NA,PB1-K-NS2/NEP-K-NA, NA-C-NS2/NEP-PB1, NA-C-PB1-NS2/NEP,NS2/NEP-PB1-C-NA, PB1-NS2/NEP-C-NA, NA-NS2/NEP-C-PB1, NA-PB1-C-NS2/NEP,NS2/NEP-C-PB1-NA, PB1-C-NS2/NEP-NA, NA-C-NS2/NEP-C-PB1, NA-C-PB1-C-NS2/NEP, NS2/NEP-C-PB1-C-NA, PB1-C-NS2/NEP-C-NA, NA-K-NS2/NEP-C-PB1,NA-K-PB1-C-NS2/NEP, NS2/NEP-C-PB1-K-NA, PB1-C-NS2/NEP-K-NA,NA-C-NS2/NEP-K-PB1, NA-C-PB1-K-NS2/NEP, NS2/NEP-K-PB1-C-NA,PB1-K-NS2/NEP-C-NA, NA-K-NS2/NEP-PB2, NA-K-PB2-NS2/NEP,NS2/NEP-PB2-K-NA, PB2-NS2/NEP-K-NA, NA-NS2/NEP-K-PB2, NA-PB2-K-NS2/NEP,NS2/NEP-K-PB2-NA, PB2-K-NS2/NEP-NA, NA-K-NS2/NEP-K-PB2,NA-K-PB2-K-NS2/NEP, NS2/NEP-K-PB2-K-NA, PB2-K-NS2/NEP-K-NA,NA-C-NS2/NEP-PB2, NA-C-PB2-NS2/NEP, NS2/NEP-PB2-C-NA, PB2-NS2/NEP-C-NA,NA-NS2/NEP-C-PB2, NA-PB2-C-NS2/NEP, NS2/NEP-C-PB2-NA, PB2-C-NS2/NEP-NA,NA-C-NS2/NEP-C-PB2, NA-C-PB2-C-NS2/NEP, NS2/NEP-C-PB2-C-NA,PB2-C-NS2/NEP-C-NA, NA-K-NS2/NEP-C-PB2, NA-K-PB2-C-NS2/NEP,NS2/NEP-C-PB2-K-NA, PB2-C-NS2/NEP-K-NA, NA-C-NS2/NEP-K-PB2,NA-C-PB2-K-NS2/NEP, NS2/NEP-K-PB2-C-NA, PB2-K-NS2/NEP-C-NA,NA-K-NS2/NEP-PB1F2, NA-K-PB1F2-NS2/NEP, NS2/NEP-PB1F2-K-NA,PB1F2-NS2/NEP-K-NA, NA-NS2/NEP-K-PB1F2, NA-PB1F2-K-NS2/NEP,NS2/NEP-K-PB1F2-NA, PB1F2-K-NS2/NEP-NA, NA-K-NS2/NEP-K-PB1F2,NA-K-PB1F2-K-NS2/NEP, NS2/NEP-K-PB1F2-K-NA, PB1F2-K-NS2/NEP-K-NA,NA-C-NS2/NEP-PB1F2, NA-C-PB1F2-NS2/NEP, NS2/NEP-PB1F2-C-NA,PB1F2-NS2/NEP-C-NA, NA-NS2/NEP-C-PB1F2, NA-PB1F2-C-NS2/NEP,NS2/NEP-C-PB1F2-NA, PB1F2-C-NS2/NEP-NA, NA-C-NS2/NEP-C-PB1F2,NA-C-PB1F2-C-NS2/NEP, NS2/NEP-C-PB1F2-C-NA, PB1F2-C-NS2/NEP-C-NA,NA-K-NS2/NEP-C-PB1F2, NA-K-PB1F2-C-NS2/NEP, NS2/NEP-C-PB1F2-K-NA,PB1F2-C-NS2/NEP-K-NA, NA-C-NS2/NEP-K-PB1F2, NA-C-PB1F2-K-NS2/NEP,NS2/NEP-K-PB1F2-C-NA, PB1F2-K-NS2/NEP-C-NA, NA-K-PA, PA-K-NA,NA-K-PA-PA, PA-PA-K-NA, NA-PA-K-PA, PA-K-PA-NA, NA-K-PA-K-PA,PA-K-PA-K-NA, NA-C-PA, PA-C-NA, NA-C-PA-PA, PA-PA-C-NA, NA-PA-C-PA,PA-C-PA-NA, NA-C-PA-C-PA, PA-C-PA-C-NA, NA-K-PA-C-PA, PA-C-PA-K-NA,NA-C-PA-K-PA, PA-K-PA-C-NA, NA-K-PA-PB1, NA-K-PB1-PA, PA-PB1-K-NA,PB1-PA-K-NA, NA-PA-K-PB1, NA-PB1-K-PA, PA-K-PB1-NA, PB1-K-PA-NA,NA-K-PA-K-PB1, NA-K-PB1-K-PA, PA-K-PB1-K-NA, PB1-K-PA-K-NA, NA-C-PA-PB1,NA-C-PB1-PA, PA-PB1-C-NA, PB1-PA-C-NA, NA-PA-C-PB1, NA-PB1-C-PA,PA-C-PB1-NA, PB1-C-PA-NA, NA-C-PA-C-PB1, NA-C-PB1-C-PA, PA-C-PB1-C-NA,PB1-C-PA-C-NA, NA-K-PA-C-PB1, NA-K-PB1-C-PA, PA-C-PB1-K-NA,PB1-C-PA-K-NA, NA-C-PA-K-PB1, NA-C-PB1-K-PA, PA-K-PB1-C-NA,PB1-K-PA-C-NA, NA-K-PA-PB2, NA-K-PB2-PA, PA-PB2-K-NA, PB2-PA-K-NA,NA-PA-K-PB2, NA-PB2-K-PA, PA-K-PB2-NA, PB2-K-PA-NA, NA-K-PA-K-PB2,NA-K-PB2-K-PA, PA-K-PB2-K-NA, PB2-K-PA-K-NA, NA-C-PA-PB2, NA-C-PB2-PA,PA-PB2-C-NA, PB2-PA-C-NA, NA-PA-C-PB2, NA-PB2-C-PA, PA-C-PB2-NA,PB2-C-PA-NA, NA-C-PA-C-PB2, NA-C-PB2-C-PA, PA-C-PB2-C-NA, PB2-C-PA-C-NA,NA-K-PA-C-PB2, NA-K-PB2-C-PA, PA-C-PB2-K-NA, PB2-C-PA-K-NA,NA-C-PA-K-PB2, NA-C-PB2-K-PA, PA-K-PB2-C-NA, PB2-K-PA-C-NA,NA-K-PA-PB1F2, NA-K-PB1F2-PA, PA-PB1F2-K-NA, PB1F2-PA-K-NA,NA-PA-K-PB1F2, NA-PB1F2-K-PA, PA-K-PB1F2-NA, PB1F2-K-PA-NA,NA-K-PA-K-PB1F2, NA-K-PB1F2-K-PA, PA-K-PB1F2-K-NA, PB1F2-K-PA-K-NA,NA-C-PA-PB1F2, NA-C-PB1F2-PA, PA-PB1F2-C-NA, PB1F2-PA-C-NA,NA-PA-C-PB1F2, NA-PB1F2-C-PA, PA-C-PB1F2-NA, PB1F2-C-PA-NA,NA-C-PA-C-PB1F2, NA-C-PB1F2-C-PA, PA-C-PB1F2-C-NA, PB1F2-C-PA-C-NA,NA-K-PA-C-PB 1F2, NA-K-PB1F2-C-PA, PA-C-PB1F2-K-NA, PB1F2-C-PA-K-NA,NA-C-PA-K-PB1F2, NA-C-PB1F2-K-PA, PA-K-PB1F2-C-NA, PB1F2-K-PA-C-NA,NA-K-PB1, PB1-K-NA, NA-K-PB1-PB1, PB1-PB1-K-NA, NA-PB1-K-PB1,PB1-K-PB1-NA, NA-K-PB1-K-PB1, PB1-K-PB1-K-NA, NA-C-PB1, PB1-C-NA,NA-C-PB1-PB1, PB1-PB1-C-NA, NA-PB1-C-PB1, PB1-C-PB1-NA, NA-C-PB1-C-PB1,PB1-C-PB1-C-NA, NA-K-PB1-C-PB1, PB1-C-PB1-K-NA, NA-C-PB1-K-PB1,PB1-K-PB1-C-NA, NA-K-PB1-PB2, NA-K-PB2-PB1, PB1-PB2-K-NA, PB2-PB1-K-NA,NA-PB1-K-PB2, NA-PB2-K-PB1, PB1-K-PB2-NA, PB2-K-PB1-NA, NA-K-PB1-K-PB2,NA-K-PB2-K-PB1, PB1-K-PB2-K-NA, PB2-K-PB1-K-NA, NA-C-PB1-PB2,NA-C-PB2-PB1, PB1-PB2-C-NA, PB2-PB1-C-NA, NA-PB1-C-PB2, NA-PB2-C-PB1,PB1-C-PB2-NA, PB2-C-PB1-NA, NA-C-PB1-C-PB2, NA-C-PB2-C-PB1,PB1-C-PB2-C-NA, PB2-C-PB1-C-NA, NA-K-PB1-C-PB2, NA-K-PB2-C-PB1,PB1-C-PB2-K-NA, PB2-C-PB1-K-NA, NA-C-PB1-K-PB2, NA-C-PB2-K-PB1,PB1-K-PB2-C-NA, PB2-K-PB1-C-NA, NA-K-PB1-PB1F2, NA-K-PB1F2-PB1,PB1-PB1F2-K-NA, PB1F2-PB1-K-NA, NA-PB1-K-PB1F2, NA-PB1F2-K-PB1,PB1-K-PB1F2-NA, PB1F2-K-PB1-NA, NA-K-PB1-K-PB1F2, NA-K-PB1F2-K-PB1,PB1-K-PB1F2-K-NA, PB1F2-K-PB1-K-NA, NA-C-PB1-PB1F2, NA-C-PB1F2-PB1,PB1-PB1F2-C-NA, PB1F2-PB1-C-NA, NA-PB1-C-PB1F2, NA-PB1F2-C-PB1,PB1-C-PB1F2-NA, PB1F2-C-PB1-NA, NA-C-PB1-C-PB1F2, NA-C-PB1F2-C-PB1,PB1-C-PB1F2-C-NA, PB1F2-C-PB1-C-NA, NA-K-PB1-C-PB1F2, NA-K-PB1F2-C-PB1,PB1-C-PB1F2-K-NA, PB1F2-C-PB1-K-NA, NA-C-PB1-K-PB1F2, NA-C-PB1F2-K-PB1,PB1-K-PB1F2-C-NA, PB1F2-K-PB1-C-NA, NA-K-PB2, PB2-K-NA, NA-K-PB2-PB2,PB2-PB2-K-NA, NA-PB2-K-PB2, PB2-K-PB2-NA, NA-K-PB2-K-PB2,PB2-K-PB2-K-NA, NA-C-PB2, PB2-C-NA, NA-C-PB2-PB2, PB2-PB2-C-NA,NA-PB2-C-PB2, PB2-C-PB2-NA, NA-C-PB2-C-PB2, PB2-C-PB2-C-NA,NA-K-PB2-C-PB2, PB2-C-PB2-K-NA, NA-C-PB2-K-PB2, PB2-K-PB2-C-NA,NA-K-PB2-PB1F2, NA-K-PB1F2-PB2, PB2-PB1F2-K-NA, PB1F2-PB2-K-NA,NA-PB2-K-PB1F2, NA-PB1F2-K-PB2, PB2-K-PB1F2-NA, PB1F2-K-PB2-NA,NA-K-PB2-K-PB1F2, NA-K-PB1F2-K-PB2, PB2-K-PB1F2-K-NA, PB1F2-K-PB2-K-NA,NA-C-PB2-PB1F2, NA-C-PB1F2-PB2, PB2-PB1F2-C-NA, PB1F2-PB2-C-NA,NA-PB2-C-PB1F2, NA-PB1F2-C-PB2, PB2-C-PB1F2-NA, PB1F2-C-PB2-NA,NA-C-PB2-C-PB1F2, NA-C-PB1F2-C-PB2, PB2-C-PB1F2-C-NA, PB1F2-C-PB2-C-NA,NA-K-PB2-C-PB1F2, NA-K-PB1F2-C-PB2, PB2-C-PB1F2-K-NA, PB1F2-C-PB2-K-NA,NA-C-PB2-K-PB1F2, NA-C-PB1F2-K-PB2, PB2-K-PB1F2-C-NA, PB1F2-K-PB2-C-NA,NA-K-PB1F2, PB1F2-K-NA, NA-K-PB1F2-PB1F2, PB1F2-PB1F2-K-NA,NA-PB1F2-K-PB1F2, PB1F2-K-PB1F2-NA, NA-K-PB1F2-K-PB1F2,PB1F2-K-PB1F2-K-NA, NA-C-PB1F2, PB1F2-C-NA, NA-C-PB1F2-PB1F2,PB1F2-PB1F2-C-NA, NA-PB1F2-C-PB1F2, PB1F2-C-PB1F2-NA,NA-C-PB1F2-C-PB1F2, PB1F2-C-PB1F2-C-NA, NA-K-PB1F2-C-PB1F2,PB1F2-C-PB1F2-K-NA, NA-C-PB1F2-K-PB1F2 or PB1F2-K-PB1F2-C-NA. Mostpreferably, the arrangement is NP-K-M1-C-HA.

It is within the scope of the present invention that every protein canbe combined with any other protein and that any two proteins can orcannot be connected or linked by either a cleavage site or a linkerpeptide.

In preferred embodiments, the expression system is for use in theprophylaxis or treatment of viral infection, particularly preferably foruse in the prophylaxis or treatment of an orthomyxovirus infection,preferably an influenza virus infection, more preferably an influenza Avirus infection, and/or in the manufacturing of medicament for use inthe prophylaxis or treatment of an orthomyxovirus infection, preferablyan influenza virus infection, more preferably an influenza A virusinfection, and/or for use in methods of prophylaxis or treatment of anorthomyxovirus infection, preferably an influenza virus infection, morepreferably an influenza A virus infection, preferably an influenza virusinfection, more preferably an influenza A virus infection.

In preferred embodiments, the expression system is for use in enhancingan immune response, preferably a B cell immune response anorthomyxovirus infection, preferably an influenza virus infection, morepreferably an influenza A virus infection.

In a preferred embodiment of this aspect, HA is defined according to theeighth aspect.

It is particularly preferred that the viral polyprotein encoded by thefirst, the second and the third polynucleotide has an amino acidaccording to SEQ ID NO: 13 or a variant thereof and/or is encoded by apolynucleotide having the nucleic acid sequence of SEQ ID NO: 14 or avariant thereof. Preferably, the encoded triple antigen proteinNP-M1-H1p is processed into a cytoplasmic NP-M1 fusion protein and amembrane spanning H1p protein by the 2A sequence.

In preferred embodiments, the expression system is for use in theprophylaxis or treatment of an orthomyxovirus infection, preferably aninfluenza A virus infection.

In more preferred embodiments, the expression system is for use inenhancing an immune response, preferably a B cell immune responseagainst a an orthomyxovirus protein, preferably an influenza A virusprotein.

In preferred embodiments, the vector or vectors comprising the first,and the second and/or the third polynucleotide is/are selected from thegroup consisting of plasmid, cosmid, phage, virus, and artificialchromosome. More preferably, a vector suitable for practicing thepresent invention is selected from the group consisting of plasmidvectors, cosmid vectors, phage vectors, preferably lambda phage andfilamentous phage vectors, viral vectors, adenovirus vectors (e.g.,non-replicating Ad5, Ad11, Ad26, Ad35, Ad49, ChAd3, ChAd4, ChAd5, ChAd6,ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20,ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd55,ChAd63, ChAd 73, ChAd82, ChAd83, ChAd146, ChAd147, PanAd1, PanAd2, andPanAd3 vectors or replication-competent Ad4 and Ad7 vectors),adeno-associated virus (AAV) vectors (e.g., AAV type 5 and type 2),alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE),sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN chimeras),herpes virus vectors (e.g. vectors derived from cytomegaloviruses, likerhesus cytomegalovirus (RhCMV) (14)), arena virus vectors (e.g.lymphocytic choriomeningitis virus (LCMV) vectors (15)), measles virusvectors, pox virus vectors (e.g., vaccinia virus, modified vacciniavirus Ankara (MVA), NYVAC (derived from the Copenhagen strain ofvaccinia), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV)vectors), vesicular stomatitis virus vectors, retrovirus, lentivirus,viral like particles, and bacterial spores. The vectors ChAd3, ChAd4,ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17,ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38,ChAd44, ChAd63 and ChAd82 are described in detail in WO 2005/071093. Thevectors PanAd1, PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAd146, andChAd147 are described in detail in WO 2010/086189. It is particularlypreferred that the vector is selected from the group consisting of MVA,ChAd63 and PanAd3.

In preferred embodiments, the expression system is for use in medicine.In more preferred embodiments, the expression system is for use in theprophylaxis or treatment of viral infection, particularly preferably foruse in the prophylaxis or treatment of RSV infection.

In a second aspect, the present invention provides an isolated proteinmixture encoded by the expression system of the first aspect.Preferably, the isolated protein mixture contains, essentially containsor comprises one or more of the viral proteins encoded by the expressionsystem of the first aspect.

In preferred embodiments, the isolated protein mixture is for use inmedicine. In particularly preferred embodiments, the isolated proteinmixture is for use in the prophylaxis or treatment of viral infection,particularly preferably for use in the prophylaxis or treatment of RSVinfection or in the prophylaxis or treatment of influenza A infection.

In a third aspect, the present invention provides an isolated host cellcontaining the expression system of the first aspect and/or the proteinmixture of the second aspect. It is understood that such host cellincludes but is not limited to prokaryotic (e.g. a bacterial cell) oreukaryotic cells (e.g. a fungal, plant or animal cell).

In preferred embodiments, the host cell is for use in medicine. Inparticularly preferred embodiments, the host cell is for use in theprophylaxis or treatment of viral infection, particularly preferably foruse in the prophylaxis or treatment of RSV infection or in theprophylaxis or treatment of influenza A infection.

In a fourth aspect, the present invention provides a compositioncomprising the expression system of the first aspect or the proteinmixture of the second aspect and a pharmaceutical acceptable carrierand/or excipient. Preferably, such composition is a pharmaceuticalcomposition.

The composition of the fourth aspect contains a therapeuticallyeffective amount of the compound, preferably in purified form, togetherwith a suitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

The compositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides.

For preparing pharmaceutical compositions of the present invention,pharmaceutically acceptable carriers can be either solid or liquid.

Solid form compositions include powders, tablets, pills, capsules,lozenges, cachets, suppositories, and dispersible granules. A solidexcipient can be one or more substances, which may also act as diluents,flavoring agents, binders, preservatives, tablet disintegrating agents,or an encapsulating material. In powders, the excipient is preferably afinely divided solid, which is in a mixture with the finely dividedinhibitor of the present invention. In tablets, the active ingredient ismixed with the carrier having the necessary binding properties insuitable proportions and compacted in the shape and size desired.Suitable excipients are magnesium carbonate, magnesium stearate, talc,sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. For preparing suppositories, a low melting wax,such as a mixture of fatty acid glycerides or cocoa butter, is firstmelted and the active component is dispersed homogeneously therein, asby stirring. The molten homogeneous mixture is then poured intoconvenient sized molds, allowed to cool, and thereby to solidify.Tablets, powders, capsules, pills, cachets, and lozenges can be used assolid dosage forms suitable for oral administration.

Liquid form composition include solutions, suspensions, and emulsions,for example, water, saline solutions, aqueous dextrose, glycerolsolutions or water/propylene glycol solutions. A saline solution is apreferred carrier when the pharmaceutical composition is administeredintravenously or intranasally by a nebulizer. For parenteral injection,liquid preparations can be formulated in solution in, e.g. aqueouspolyethylene glycol solution.

In a particularly preferred embodiment of this aspect, thepharmaceutical composition is in the form of a solution, suspension, oremulsion and is administered intranasally by a nebulizer.

Preferably, the pharmaceutical composition is in unit dosage form. Insuch form the composition may be subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged composition, the package containing discrete quantities ofthe composition, such as packeted tablets, capsules, and powders invials or ampoules. Also, the unit dosage form can be a capsule, aninjection vial, a tablet, a cachet, or a lozenge itself, or it can bethe appropriate number of any of these in packaged form.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents.

Furthermore, such composition may also comprise other pharmacologicallyactive substance such as but not limited to adjuvants and/or additionalactive ingredients.

Adjuvants in the context of the present invention include but are notlimited to Examples of such adjuvants include but are not limited toinorganic adjuvants, organic adjuvants, oil-based adjuvants, cytokines,particulate adjuvants, virosomes, bacterial adjuvants, syntheticadjuvants, or synthetic polynucleotides adjuvants.

Additional active ingredients include but are not limited to othervaccine compounds or compositions. Preferably, the additional activeingredient is another viral vaccine, more preferably a vaccine against aDNA virus, a negative sense single stranded (ssRNA(−)) RNA virus or anambisense RNA virus. Further preferred, the virus is selected fromnegative-single stranded (ssRNA(−)) RNA virus. Even more preferred, thevirus is selected from enveloped ssRNA(−) viruses, more preferably fromthe group consisting of paramyxoviruses and orthomyxoviruses.Preferably, the additional active ingredient is a vaccine againstparamyxoviruses, preferably selected from the group consisting ofPneumovirinae, Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus,Salem-Virus, Tupaia-Paramyxovirus, Beilong-Virus, J-Virus,Menangle-Virus, Mossmann-Virus, and Murayama-Virus. It is particularlypreferred that the Pneumovirinae is selected from the group consistingof Pneumovirus, (e.g. human respiratory syncytical virus (RSV), murinepneumonia virus, bovine RSV, ovine RSV, caprine RSV) andMetapneumovirus, (e.g. human metapneumovirus, avaian metapneumovirus).It is particularly preferred that the Paramyxovirinae is selected fromthe group consisting of Respirovirus (e.g. human parainfluenza virus 1and 3), and Rubulavirus, (e.g. human parainfluenza virus 2 and 4).Alternatively or additionally, the additional active ingredient ispreferably another viral vaccine against an orthomyxovirus, morepreferably selected from the genus of Influenzavirus A, InfluenzavirusB, Influenzavirus C, Thogotoviris and Isavirus. In even more preferredembodiments, the orthomxyovirus is Influenzavirus A, preferably selectedfrom the influenza A virus subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8,H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, H10N7,more preferably the influenza A virus subtype H1N1.

In a fifth aspect the present invention provides for an expressionsystem of the first aspect, the isolated protein mixture of the secondaspect, the isolated host cell of the third aspect or the composition ofthe fourth aspect, for the use in the treatment or prevention of a viraldisease.

In preferred embodiments of this aspect, the viral disease is caused bya DNA virus, a negative sense single stranded (ssRNA(−)) RNA virus or anambisense RNA virus. Further preferred, the virus is selected fromnegative-single stranded (ssRNA(−)) RNA virus. Even more preferred, thevirus is selected from enveloped ssRNA(−) viruses, more preferably fromthe group consisting of paramyxoviruses and orthomyxoviruses. Theparamyxovirus is preferably selected from the group consisting ofPneumovirinae, Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus,Salem-Virus, Tupaia-Paramyxovirus, Beilong-Virus, J-Virus,Menangle-Virus, Mossmann-Virus, and Murayama-Virus. Even morepreferably, the Pneumovirinae is selected from the group consisting ofPneumovirus, (e.g. human respiratory syncytical virus (RSV), murinepneumonia virus, bovine RSV, ovine RSV, caprine RSV) andMetapneumovirus, (e.g. human metapneumovirus, avaian metapneumovirus).Even more preferably, the Paramyxovirinae is selected from the groupconsisting of Respirovirus (e.g. human parainfluenza virus 1 and 3), andRubulavirus, (e.g. human parainfluenza virus 2 and 4). Alternatively oradditionally, the viral disease is preferably caused by anorthomyxovirus, more preferably selected from the genus ofInfluenzavirus A, Influenzavirus B, Influenzavirus C, Thogotoviris andIsavirus. In even more preferred embodiments, the orthomxyovirus isInfluenzavirus A, preferably selected from the influenza A virussubtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8,H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, H10N7, more preferably theinfluenza A virus subtype H1N1.

In a sixth aspect, the present invention provides for a method oftreatment or prevention of a viral disease comprising the administrationof effective amounts of the expression system of the first aspect, theisolated protein mixture of the second aspect, the isolated host cell ofthe third aspect or the composition of the fourth aspect for the use inthe treatment or prevention of a viral disease.

In preferred embodiments of this aspect, the viral disease is caused bya DNA virus, a negative sense single stranded (ssRNA(−)) RNA virus or anambisense RNA virus. Further preferred, the virus is selected fromnegative-single stranded (ssRNA(−)) RNA virus. Even more preferred, thevirus is selected from enveloped ssRNA(−) viruses, more preferably fromthe group consisting of paramyxoviruses and orthomyxoviruses. Theparamyxovirus is preferably selected from the group consisting ofPneumovirinae, Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus,Salem-Virus, Tupaia-Paramyxovirus, Beilong-Virus, J-Virus,Menangle-Virus, Mossmann-Virus, and Murayama-Virus. Even morepreferably, the Pneumovirinae is selected from the group consisting ofPneumovirus, (e.g. human respiratory syncytical virus (RSV), murinepneumonia virus, bovine RSV, ovine RSV, caprine RSV) andMetapneumovirus, (e.g. human metapneumovirus, avaian metapneumovirus).Even more preferably, the Paramyxovirinae is selected from the groupconsisting of Respirovirus (e.g. human parainfluenza virus 1 and 3), andRubulavirus, (e.g. human parainfluenza virus 2 and 4). Alternatively oradditionally, the viral disease is preferably caused by anorthomyxovirus, more preferably selected from the genus ofInfluenzavirus A, Influenzavirus B, Influenzavirus C, Thogotoviris andIsavirus. In even more preferred embodiments, the orthomxyovirus isInfluenzavirus A, preferably selected from the influenza A virussubtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8,H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, H10N7, more preferably theinfluenza A virus subtype H1N1.

In a seventh aspect, the present invention provides for a method ofenhancing an immune response against an immunogen comprising theadministration of the expression system of the first aspect, the proteinmixture of the second aspect, the cell of the third aspect and thecomposition of the fourth aspect.

In preferred embodiments of this aspect, the immunogen is a pathogen,more preferred the immunogen is a virus. Preferably, the virus isselected from the group consisting of a DNA virus, a negative sensesingle stranded (ssRNA(−)) RNA virus or an ambisense RNA virus. Furtherpreferred, the virus is selected from negative-single stranded(ssRNA(−)) RNA virus. Even more preferred, the virus is selected fromenveloped ssRNA(−) viruses, more preferably from the group consisting ofparamyxoviruses and orthomyxoviruses. The paramyxovirus is preferablyselected from the group consisting of Pneumovirinae, Paramyxovirinae,Fer-de-Lance-Virus, Nariva-Virus, Salem-Virus, Tupaia-Paramyxovirus,Beilong-Virus, J-Virus, Menangle-Virus, Mossmann-Virus, andMurayama-Virus. Even more preferably, the Pneumovirinae is selected fromthe group consisting of Pneumovirus, (e.g. human respiratory syncyticalvirus (RSV), murine pneumonia virus, bovine RSV, ovine RSV, caprine RSV)and Metapneumovirus, (e.g. human metapneumovirus, avaianmetapneumovirus). Even more preferably, the Paramyxovirinae is selectedfrom the group consisting of Respirovirus (e.g. human parainfluenzavirus 1 and 3), and Rubulavirus, (e.g. human parainfluenza virus 2 and4). Alternatively or additionally, the viral disease is preferablycaused by an orthomyxovirus, more preferably selected from the genus ofInfluenzavirus A, Influenzavirus B, Influenzavirus C, Thogotoviris andIsavirus. In even more preferred embodiments, the orthomxyovirus isInfluenzavirus A, preferably selected from the influenza A virussubtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8,H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, H10N7, more preferably theinfluenza A virus subtype H1N1.

In an eighth aspect, the present invention provides nucleotideconstructs encoding influenza hemagglutinin (HA), an expression systemcomprising these nucleotide constructs, and proteins or polyproteinsencoded by the nucleotide constructs, wherein the HA0 cleavage site hasa multibasic sequence.

The nucleic acid construct of this aspect comprises, essentiallyconsists or consists of a polynucleotide encoding a modified influenzahemagglutinin (HA), wherein the HA0 cleavage site is modified byintroducing one or more basic amino acids. Preferably, in the modifiedHA, the HA0 cleavage site of the consensus HA gene was substituted withthe multibasic cleavage site of H5N1 that is cleaved by ubiquitousproteases to obtain a fully processed HA (H1p).

Influenza hemagglutinin (HA) is a protein belonging to the group ofviral hemagglutinins found on the surface of the influenza viruses. Itis an antigenic glycoprotein which is responsible for binding the virusto the cell that is being infected. HA proteins like influenzahemagglutinin bind to cells with sialic acid on the membranes, such ascells in the upper respiratory tract or erythrocytes. There are at least16 different HA antigens. These serotypes or subtypes are named H1through H16.

HA has two functions:

1. Recognition of target cells by binding to sialic acid-containingreceptors.

2. Mediating the entry of the viral genome into the target cells bycausing the fusion of host endosomal membrane with the viral membrane(envelop) In detail, HA binds to the monosaccharide sialic acid which ispresent on the surface of its target cells. The cell membrane thenengulfs the virus and the portion of the membrane that encloses thevirus forms an endosome. Then the endosome is acidified and beingtransformed it into a lysosome. As soon as the pH within the endosomedrops to about 6.0, the original folded structure of the HA moleculebecomes unstable, causing it to partially unfold, releasing ahydrophobic portion of its peptide chain. This fusion peptide actsinserts into the endosomal membrane. Then, the rest of the HA moleculerefolds into a new structure and causes the fusion of the viral membranewith the endosomal membrane such that the contents of the virus,including its RNA genome, are released into the cytoplasm of the cell.

To acquire its membrane fusion potential, HA0 must be cleaved into HA1and HA2 by host cell proteases. Cleavage occurs at a linker sequenceconnecting the HA1 and HA2 subunits, which is located on a partiallysurface exposed loop. The H0 cleavage site of the influenza HA islocated at about aa 340 in the H1N1 subtype (aa 339 to 344 of SEQ ID NO:8), the H5N1 subtype (aa 337 to 346 of SEQ ID NO: 10) and H3N2 (aa 340to 350 of SEQ ID NO: 20). The position of the H0 cleavage site in othersubtypes of Influenza virus HA can be determined by the skilled personby conducting sequence alignments and analysing the homology of thesequences by methods well-known in this technical field. Influenza Avirus HA of subtype H1N1 and H3N2 require cleavage by host cellproteases to transit into a fusion-competent state. Proteolyticactivation of influenza viruses can occur in the Golgi apparatus or atthe plasma membrane of infected cells, as well as in the extracellularspace and in target cell vesicles, so the nature of the cleavage siteand the respective activating proteases have important implications forthe biological properties of influenza virus as well as for therapeuticintervention. HA of subtype H5N1 were shown to harbour several arginineand lysine residues at the cleavage site, with an R-X-R/K-R consensussequence being indispensable for efficient cleavage. In addition,evidence was obtained that cleavage of HA might occur in the trans-Golginetwork (TGN). It has been demonstrated that these viruses are activatedby furin.

The amino acid sequence of SEQ ID NO: 8 is a consensus sequence derivedfrom the alignment of 829 sequences of the H1N1 subtype annotated in theNCBI Influenza Virus Resource Database, circulating worldwide from Aprilto September 2009. The amino acid sequence of SEQ ID NO: 9 is identicalto SEQ ID NO: 8 with the exception that the natural H0 protease cleavagesite has been substituted with a multibasic site derived from H5N1. Theamino acid sequence of SEQ ID NO: 10 is a consensus derived from thealignment of 259 sequences of the H5N1 subtype annotated in the NCBIInfluenza Virus Resource Database, infecting humans worldwide from 1990to 2009. The amino acid sequence SEQ ID NO: 20 is the sequence of HA ofinfluenza A virus subtype H3N2, strain A/Wellington/01/2004(H3N2). Theamino acid sequence of SEQ ID NO: 21 is based on SEQ ID NO: 20 whereinthe natural H0 protease cleavage site has been substituted with amultibasic site derived from H5N1

The amino acid sequence of SEQ ID NO: 11 is a NP consensus sequencewhich was designed on the basis of the alignment of the differentinfluenza subtype consensus sequences. Further, the NP sequence of SEQID NO: 11 lacks the Nuclear Localization Signal residing in aa 6-8 (TRKto AAA) to increase cytoplasmic expression.

The amino acid sequence of SEQ ID NO: 12 is a M1 consensus sequencewhich was derived by alignment of different consensus sequences whichwere aligned and the most common amino acid at each position was chosen.

In a preferred embodiment of this aspect, the nucleic acid constructand/or the expression system comprising this nucleic acid construct,comprises elements to direct transcription and translation of the HA andthe optional further proteins encoded by the nucleic acid constructand/or the expression system, which may be included in the preferredembodiments outlined below. Such elements included promoter and enhancerelements to direct transcription of mRNA in a cell-free or a cell-basedbased system, preferably a cell-based system. In another embodiment,wherein the polynucleotides are provided as translatable RNAs isenvisioned that the expression system comprises those elements that arenecessary for translation and/or stabilization of RNAs encoding the HAand/or the T cell inducing protein(s), e.g. polyA-tail, IRES, capstructures etc.

In a preferred embodiment of this aspect, the nucleic acid constructencodes a HA protein, peptide or variant thereof comprising a modifiedH0 cleavage site, wherein the HA is selected from the group of HAsubtypes consisting of H1, H2, H3, H4, H6, H7, H8, H9, H10, H11, H12,H13, H14, H15, H16 or a variant thereof or a consensus sequence thereof,or a variant thereof or a consensus sequence of one or more of the HAsubtypes selected from the group of HA subtypes consisting of H1, H2,H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16.Preferably, the HA subtypes are selected from the group consisting ofH1, H2, H3, H7, H9, H10, or a variant thereof or a consensus sequencethereof, or a variant thereof or a consensus sequence of one or more ofthe HA subtypes selected from the group of HA subtypes consisting of H1,H2, H3, H5, H7, H9, H10. More preferred, the HA protein, peptide orvariant thereof which comprises a modified H0 cleavage site, is a HAfrom subtype H1 or a variant thereof. Most preferred, the polynucleotideencoding the HA of the nucleotide construct has the sequence of SEQ IDNO: 9.

In a preferred embodiment of this aspect, the nucleic acid constructencodes a HA protein, peptide or variant thereof, wherein H0 cleavagesite is modified by substituting at least one non-basic amino acid by abasic amino acid and/or by introducing at least one basic amino acidinto the sequence of the H0 cleavage site. Preferably, the basic aminoacid is selected from the group consisting of arginine (Arg; R), lysine(Lys; K) and histidine (His, H). More preferably, the basic amino acidis selected from the group consisting of arginine (Arg; R) and lysine(Lys; K).

Preferably, the cleavage site comprises a sequence of 6 to 12 aminoacids, more preferably 10-12 amino-acids.

Preferably, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or 100% to theamino acids of the polypeptide forming the H0 cleavage site are basicamino acids.

Preferably, the HA0 cleavage site of has a sequence selected from thegroup consisting of PQRERRRKKR (SEQ ID NO: 15), PQRESRRKKR (SEQ ID NO:16), PQGERRRKKR (SEQ ID NO: 17), PLRERRRKR (SEQ ID NO: 18) and PQRETR(SEQ ID NO: 19). Most preferred, the HA0 cleavage site of has thesequence PQRERRRKKR (SEQ ID NO: 15).

In Table 1 the sequences of the H0 cleavage site of H1N1 strain and thesequences of the H0 cleavage site of H5N1 strain are compared in theirrespective context of the HA amino acid chain. In the first line, theconsensus sequence of the H0 polybasic cleavage site of the highlypathogenic H5N1 subtype is indicated. In the second line, the amino acidsequence of the H0 cleavage site of the HA of the H1N1 wild-type strainis indicated. In the third line the engineered sequence of the polybasiccleavage site in the H1p protein, replacing the natural sequence.

TABLE 1 H5N1 cleavage LATGLRNS PQRERRRKKR GLFGAIA . . . siteH1N1 cleavage LATGLRNV PSIQSR     GLFGAIA . . . site H1p cleavageLATGLRNI PQRERRRKKR GLFGAIA . . . site

In embodiments of the eighth aspect of the present invention, thenucleic acid construct is part of an expression system encoding themodified HA and a second polynucleotide. In this expression system thepolynucleotide encoding the modified HA and the second polynucleotideare comprised on separate vectors or on the same vector. Accordingly,the polynucleotide encoding the modified HA may be comprised on onevector and the second polynucleotide may be comprised on a secondvector. Alternatively or additionally, the polynucleotide encoding themodified HA and the second polynucleotide may be comprised on the samevector. It is preferred that the polynucleotide encoding the modified HAand the second polynucleotide are comprised on the same vector. It isparticularly preferred that the polynucleotide encoding the modified HAand the second polynucleotide comprised on the same vector are linked insuch that they are expressed as a polyprotein. Preferably, thepolynucleotide encoding the modified HA and the second polynucleotideform an open reading frame.

It is preferred that the polynucleotide encoding the modified HA and thesecond polynucleotide are expressed as an artificial polyprotein. In thecontext of the present invention the term “artificial polyprotein” isdirected at polyproteins which are not naturally occurring, e.g. whichare generated by using recombinant DNA techniques. Accordingly, theproteins, peptides or variants thereof encoded in this artificialpolyprotein are preferably derived from pathogens which genome do notencode a polyprotein comprising the proteins, peptides or variantsencoded by the polynucleotide encoding the modified HA and secondpolynucleotide of the invention. Preferably, the polynucleotide encodingthe modified HA and the second polynucleotide are both derived frominfluenza A viruses.

In preferred embodiments of the eighth aspect, the second polynucleotideencodes a protein or variant thereof, which induces a T cell response,and which is, preferably, a non-structural and/or internal protein ofinfluenza A virus. Preferably, the non-structural and/or internalprotein encoded by the second polynucleotide is selected from the groupconsisting of NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2).

It is preferred that the amino acid sequence of the modified HA and/orthe non-structural (internal) protein encoded by the secondpolynucleotide comprises consecutive segments or a consensus sequence ofone or more different virus isolates.

In the context of the present invention it is preferred that the term“segment” refers to a part of a protein or polyprotein. It isparticularly preferred that such segment folds and/or functionsindependently of the rest of the protein or polyprotein such as but notlimited to a domain, an epitope or a fragment thereof. It is understoodthat a protein variant in the context of the present invention differsin comparison to its parent polypeptide in changes in the amino acidsequence such as amino acid exchanges, insertions, deletions, N-terminaltruncations, or C-terminal truncations, or any combination of thesechanges, which may occur at one or several sites whereby the variantexhibits at least 80% sequence identity to its parent polypeptide.

In a further preferred embodiment, a membrane attachment domain of themodified HA or a variant thereof is functionally deleted, thus, eitherbeing structurally deleted or structurally present but not fulfillingits biological function. In a particularly preferred embodiment, theamino acid sequence corresponding to the membrane attachment domain isdeleted. The deletion of the membrane attachment region serves thepurpose of ascertaining that the anti-pathogenic B cell responseinducing protein is secreted from the cell into which the expressionsystem of the invention has been introduced.

In a further preferred embodiment of this aspect the modified HAcomprises a secretion signal, which targets the protein to theendoplasmatic reticulum (ER). Such secretion signals are presentpreferably in the context of a deleted membrane attachment domain. Theskilled person is well aware of various such secretion signals, whichmay be used as heterologous secretion signals, e.g. added to theN-terminus of the modified HA. Alternatively or additionally a naturallyoccurring secretion signal may be used, which is, e.g., present in themajority of structural and/or surface viral proteins. Thus, if naturallypresent in the respective protein, it is preferred that the secretionsignal is maintained in a modified version of the structural and/orsurface protein.

In embodiments of the eighth aspect, the non-structural protein is aconserved internal protein suitable for inducing a T cell mediatedimmune response against the pathogen involving the activation ofantigen-specific T lymphocyte such as but not limited to cytotoxic Tcells (CTLs), T helper cells (T_(H) cells), central memory T cells (TCMcells), effector memory T cells (TEM cells), and regulatory T cells(Treg cells). Thus, preferably the T cell inducing protein of thepathogen does not comprise a secretion signal.

In the context of the present invention, the modified HA or variantthereof is located either N- or C-terminally with respect to theprotein, peptide or variant thereof encoded by the secondpolynucleotide. In a preferred embodiment, the protein, peptide orvariant thereof encoded by the second polynucleotide is locatedN-terminally with respect to the modified HA or variant thereof.

In preferred embodiments of the eighth aspect, a polynucleotide encodinga cleavage site is positioned between the modified HA or variant thereofand the second polynucleotide. It is within the scope of the presentinvention that that any two proteins can or cannot be connected orlinked by a cleavage site.

It is preferred that this cleavage site is either a self-cleaving site(i.e. a cleavage site within the amino acid sequence where this sequenceis cleaved or is cleavable without such cleavage involving anyadditional molecule or where the peptide-bond formation in this sequenceis prevented in the first place) or an endopeptidase cleavage site (i.e.a cleavage cite within the amino acid sequence where this sequence iscleaved or is cleavable by an endopeptidase, e.g. trypsin, pepsin,elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8,cathepsins). More preferably, the self-cleaving site is a 2A cleavagesite selected from the group consisting of a viral 2A peptide or 2A-likepeptide of Picornavirus, insect viruses, Aphtoviridae, Rotaviruses andTrypanosoma, preferably wherein the 2A cleavage site is the 2 A peptideof foot and mouth disease virus. Alternatively or additionally, thepolyprotein of the present invention can be cleaved by an autoprotease,i.e. a protease which cleaves peptide bonds in the same protein moleculewhich also comprises the protease. Examples of such autoproteases arethe NS2 protease from flaviviruses or the VP4 protease of birnaviruses

In the context of the present invention, the cleavage site can bepositioned N-terminally with respect to the modified HA or variantthereof and C-terminally with respect to the protein, peptide or variantthereof encoded by the second polynucleotide. Alternatively the cleavagesite can be positioned C-terminally with respect to the modified HA orvariant thereof and N-terminally with respect to the protein, peptide orvariant thereof encoded by the second polynucleotide.

In preferred embodiment of the eighth aspect, the expression systemfurther comprises a third polynucleotide encoding a protein, peptide ora variant thereof of a pathogen.

It is preferred that the protein, peptide or variant thereof encoded bythe third polynucleotide differs from the modified HA or variant thereofor from the protein, peptide or variant thereof encoded by the secondpolynucleotide. Preferably, the proteins, peptides or variants thereofencoded by the first, second and the third polynucleotide differ fromeach other in that they comprise amino acid sequences of differentproteins.

In preferred embodiments of the eighth aspect, the third polynucleotideencodes a protein or variant thereof, which induces a T cell response,and which is, preferably, a non-structural and/or internal protein ofinfluenza A virus. Preferably, the non-structural and/or internalprotein encoded by the third polynucleotide is selected from the groupconsisting of NP, M1, M2, NS1, NS2/NEP, PA, PB1, PB2 or PB1-F2 (PB1F2).

In preferred embodiments a polynucleotide encoding a linker ispositioned between the second polynucleotide and the thirdpolynucleotide. It is preferred that the linker is a flexible linker,preferably a flexible linker comprising an amino acid sequence accordingto SEQ ID NO: 6 (Gly-Gly-Gly-Ser-Gly-Gly-Gly).

In preferred embodiments the third polynucleotide is comprised on aseparate or on the same vector as the polynucleotide encoding themodified HA or variant thereof and/or the second polynucleotide.

Accordingly, the polynucleotide encoding the modified HA or variantthereof is comprised on one vector and the second polynucleotide iscomprised on a second vector and the third polynucleotide is comprisedon a third vector. Alternatively or additionally, the polynucleotideencoding the modified HA or variant thereof and the secondpolynucleotide are comprised on the same vector and the thirdpolynucleotide is comprised on a separate vector, or the polynucleotideencoding the modified HA or variant thereof and the third polynucleotideare comprised on the same vector and the second polynucleotide iscomprised on a separate vector, or the second and the thirdpolynucleotide are comprised on the same vector and the polynucleotideencoding the modified HA or variant thereof is comprised on a separatevector. Alternatively or additionally, the polynucleotide encoding themodified HA or variant thereof and the second and the thirdpolynucleotide are comprised on the same vector. It is preferred thatthe polynucleotide encoding the modified HA or variant thereof and thesecond and the third polynucleotide may be comprised on the same vector.It is particularly preferred that the polynucleotide encoding themodified HA or variant thereof and the second and the thirdpolynucleotide comprised on the same vector are linked in such that theyare expressed as a polyprotein. Preferably, the polynucleotide encodingthe modified HA or variant thereof and the second and the thirdpolynucleotide comprised on the same vector form an open reading frame.

In preferred embodiments of this aspect, the vector or vectorscomprising the polynucleotide encoding the modified HA or variantthereof, and the second and/or the third polynucleotide is/are selectedfrom the group consisting of plasmid, cosmid, phage, virus, andartificial chromosome. More preferably, a vector suitable for practicingthe present invention is selected from the group consisting of plasmidvectors, cosmid vectors, phage vectors, preferably lambda phage andfilamentous phage vectors, viral vectors, adenovirus vectors (e.g.,non-replicating Ad5, Ad11, Ad26, Ad35, Ad49, ChAd3, ChAd4, ChAd5, ChAd6,ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20,ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd55,ChAd63, ChAd 73, ChAd82, ChAd83, ChAd146, ChAd147, PanAd1, PanAd2, andPanAd3 vectors or replication-competent Ad4 and Ad7 vectors),adeno-associated virus (AAV) vectors (e.g., AAV type 5 and type 2),alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE),sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN chimeras),herpes virus vectors (e.g. vectors derived from cytomegaloviruses, likerhesus cytomegalovirus (RhCMV) (14)), arena virus vectors (e.g.lymphocytic choriomeningitis virus (LCMV) vectors (15)), measles virusvectors, pox virus vectors (e.g., vaccinia virus, modified vacciniavirus Ankara (MVA), NYVAC (derived from the Copenhagen strain ofvaccinia), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV)vectors), vesicular stomatitis virus vectors, retrovirus, lentivirus,viral like particles, and bacterial spores. The vectors ChAd3, ChAd4,ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17,ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38,ChAd44, ChAd63 and ChAd82 are described in detail in WO 2005/071093. Thevectors PanAd1, PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAd146, andChAd147 are described in detail in WO 2010/086189. It is particularlypreferred that the vector is selected from the group consisting of MVA,ChAd63 and PanAd3.

In preferred embodiments of this aspect, the nucleotide construct or theexpression system or the vector or vectors comprising the polynucleotideof the nucleotide construct or the expression system may encompass“expression control sequences” that regulate the expression of the geneof interest. Typically, expression control sequences are polypeptides orpolynucleotides such as but not limited to promoters, enhancers,silencers, insulators, or repressors.

In a particularly preferred embodiment of this aspect, the expressionsystem is defined according to the embodiments of the first aspect ofthe present invention directed at expressing systems comprisingpolynucleotides encoding proteins, peptides or variants thereof fromorthomyxovirus, preferably proteins, peptides or variants from influenzaA viruses.

In preferred embodiments, the nucleic acid construct and/or theexpression system of the eighth aspect is for use in medicine. In morepreferred embodiments, the nucleic acid constructs, the expressionsystems or the proteins of this aspect are for use in the prophylaxis ortreatment of an influenza A virus infection and/or in the manufacturingof medicament for use in the prophylaxis or treatment of an influenza Avirus infection and/or for use in methods of prophylaxis or treatment ofan influenza A virus infection.

In preferred embodiments the expression system is for use in enhancingan immune response. In more preferred embodiments, the expression systemis for use in enhancing an anti-pathogenic B cell immune responseagainst an influenza A virus infection, more preferably an influenza Avirus as defined in the first aspect of the invention.

In a ninth aspect, the present invention provides the use of themultibasic HA0 cleavage site as defined in the eighth aspect forconstructing a nucleic acid construct or an expression systems capableof expressing the modified influenza hemagglutinin (HA) of the eighthaspect in vitro and/or in vivo. Furthermore, this aspect provides theisolated protein mixture, the protein and/or polyprotein encoded by thenucleic acid construct or expression system constructed according tothis aspect.

In a tenth aspect, the invention provides an isolated protein mixtureencoded by the expression system of the eighth aspect. Preferably, theisolated protein mixture contains, essentially contains or comprises oneor more of the proteins or polyproteins encoded by the nucleic acidconstruct or the expression system of the eighth aspect. In preferredembodiments, the isolated protein mixture is for use in medicine. Inparticularly preferred embodiments, the isolated protein mixture is foruse in the prophylaxis or treatment of a viral infection, particularlypreferably for use in the prophylaxis or treatment of an influenza Avirus infection and/or in the manufacturing of medicament for use in theprophylaxis or treatment of an influenza A virus infection and/or foruse in methods of prophylaxis or treatment of an influenza A virusinfection.

In an eleventh aspect, the invention provides an isolated host cellcontaining the nucleotide constructs, the expression system or theproteins or polyproteins of the eighth aspect and/or the protein mixtureof the tenth aspect. It is understood that such host cell includes butis not limited to prokaryotic (e.g. a bacterial cell) or eukaryoticcells (e.g. a fungal, plant or animal cell). In preferred embodiments ofthis aspect, the host cell is for use in medicine. In particularlypreferred embodiments, the host cell is for use in the prophylaxis ortreatment of an influenza A virus infection and/or in the manufacturingof medicament for use in the prophylaxis or treatment of an influenza Avirus infection and/or for use in methods of prophylaxis or treatment ofan influenza A virus infection.

In a twelfth aspect, the present invention provides a compositioncomprising the nucleotide constructs, the expression system or theproteins or polyproteins of the eighth aspect, or the protein mixture ofthe tenth aspect, and a pharmaceutical acceptable carrier and/orexcipient. Preferably, such composition is a pharmaceutical composition.

The composition of the twelfth aspect contains a therapeuticallyeffective amount of the compound, preferably in purified form, togetherwith a suitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

The compositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides.

For preparing pharmaceutical compositions of the present invention,pharmaceutically acceptable carriers can be either solid or liquid.

Solid form compositions include powders, tablets, pills, capsules,lozenges, cachets, suppositories, and dispersible granules. A solidexcipient can be one or more substances, which may also act as diluents,flavoring agents, binders, preservatives, tablet disintegrating agents,or an encapsulating material. In powders, the excipient is preferably afinely divided solid, which is in a mixture with the finely dividedinhibitor of the present invention. In tablets, the active ingredient ismixed with the carrier having the necessary binding properties insuitable proportions and compacted in the shape and size desired.Suitable excipients are magnesium carbonate, magnesium stearate, talc,sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. For preparing suppositories, a low melting wax,such as a mixture of fatty acid glycerides or cocoa butter, is firstmelted and the active component is dispersed homogeneously therein, asby stirring. The molten homogeneous mixture is then poured intoconvenient sized molds, allowed to cool, and thereby to solidify.Tablets, powders, capsules, pills, cachets, and lozenges can be used assolid dosage forms suitable for oral administration.

Liquid form composition include solutions, suspensions, and emulsions,for example, water, saline solutions, aqueous dextrose, glycerolsolutions or water/propylene glycol solutions. A saline solution is apreferred carrier when the pharmaceutical composition is administeredintravenously or intranasally by a nebulizer. For parenteral injection,liquid preparations can be formulated in solution in, e.g. aqueouspolyethylene glycol solution.

In a particularly preferred embodiment of this aspect, thepharmaceutical composition is in the form of a solution, suspension, oremulsion and is administered intranasally by a nebulizer.

Preferably, the pharmaceutical composition is in unit dosage form. Insuch form the composition may be subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged composition, the package containing discrete quantities ofthe composition, such as packeted tablets, capsules, and powders invials or ampoules. Also, the unit dosage form can be a capsule, aninjection vial, a tablet, a cachet, or a lozenge itself, or it can bethe appropriate number of any of these in packaged form.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents.

Furthermore, such composition may also comprise other pharmacologicallyactive substance such as but not limited to adjuvants and/or additionalactive ingredients.

Adjuvants in the context of the present invention include but are notlimited to Examples of such adjuvants include but are not limited toinorganic adjuvants, organic adjuvants, oil-based adjuvants, cytokines,particulate adjuvants, virosomes, bacterial adjuvants, syntheticadjuvants, or synthetic polynucleotides adjuvants.

Additional active ingredients include but are not limited to othervaccine compounds or compositions. Preferably, the additional activeingredient is another viral vaccine, more preferably a vaccine against aDNA virus, a negative sense single stranded (ssRNA(−)) RNA virus or anambisense RNA virus. Further preferred, the virus is selected fromnegative-single stranded (ssRNA(−)) RNA virus. Even more preferred, thevirus is selected from enveloped ssRNA(−) viruses, more preferably fromthe group consisting of paramyxoviruses and orthomyxoviruses.Preferably, the additional active ingredient is a vaccine againstparamyxoviruses, preferably selected from the group consisting ofPneumovirinae, Paramyxovirinae, Fer-de-Lance-Virus, Nariva-Virus,Salem-Virus, Tupaia-Paramyxovirus, Beilong-Virus, J-Virus,Menangle-Virus, Mossmann-Virus, and Murayama-Virus. It is particularlypreferred that the Pneumovirinae is selected from the group consistingof Pneumovirus, (e.g. human respiratory syncytical virus (RSV), murinepneumonia virus, bovine RSV, ovine RSV, caprine RSV) andMetapneumovirus, (e.g. human metapneumovirus, avaian metapneumovirus).It is particularly preferred that the Paramyxovirinae is selected fromthe group consisting of Respirovirus (e.g. human parainfluenza virus 1and 3), and Rubulavirus, (e.g. human parainfluenza virus 2 and 4).Alternatively or additionally, the additional active ingredient ispreferably another viral vaccine against an orthomyxovirus, morepreferably selected from the genus of Influenzavirus A, InfluenzavirusB, Influenzavirus C, Thogotoviris and Isavirus. In even more preferredembodiments, the orthomxyovirus is Influenzavirus A, preferably selectedfrom the influenza A virus subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8,H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, H10N7,more preferably the influenza A virus subtype H1N1.

In a thirteenth aspect, the present invention provides the nucleotideconstructs, the expression system or the proteins or polyproteins of theeighth aspect, the protein mixture of the tenth aspect, the cell of theeleventh aspect and the composition of the twelfth aspect, for the usein medicine in particular in the treatment or prevention of influenza Avirus infections. The influenza A virus is preferably selected from theinfluenza A virus subtypes H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1,H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, H10N7, morepreferably the influenza A virus subtype H1N1.

In a fourteenth aspect, the present invention provides for a method oftreatment or prevention of an influenza A virus infections comprisingthe administration of an effective amount of the nucleotide constructs,the expression system or the proteins or polyproteins of the eighthaspect, the protein mixture of the tenth aspect, the cell of theeleventh aspect and the composition of the twelfth aspect. The influenzaA virus is preferably selected from the influenza A virus subtypes H1N1,H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2,H7N3, H7N4, H7N7, H9N2, H10N7, more preferably the influenza A virussubtype H1N1.

In a fifteenth aspect, the present invention provides for a method ofenhancing an immune response comprising the administration of thenucleotide constructs or the expression system or the proteins orpolyproteins of the eighth aspect, the protein mixture of the tenthaspect, the cell of the eleventh aspect and the composition of thetwelfth aspect. In a preferred embodiment of this aspect, the methodenhances an immune response against influenza A virus. The influenza Avirus is preferably selected from the influenza A virus subtypes H1N1,H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2,H7N3, H7N4, H7N7, H9N2, H10N7, more preferably the influenza A virussubtype H1N1.

The following examples are merely illustrative of the present inventionand should not be construed to limit the scope of the invention asindicated by the appended claims in any way.

EXAMPLES Example 1 Design and Synthesis of DNA Encoding Optimised RSVAntigen

Consensus Vaccine

Several computational alternatives to isolate-based vaccine designexist. One approach is reconstruction of the most recent common ancestor(MRCA) sequence (9). In this type of analysis, the ancestral state is anestimate of the actual sequence that existed in the past (i.e., it comesdirectly from the reconstructed history). Another type of computationalanalysis is a center of the tree (COT) approach. The COT approachidentifies a point on the unrooted phylogeny, where the averageevolutionary distance from that point to each tip on the phylogeny isminimized. Advocates of this approach state that because the COT is apoint on the phylogeny, the estimated COT sequence will have the sameadvantages as the estimated ancestral sequence. See, for example, U.S.Application 2005/0137387 A1. However, this COT approach is sufficientlycomplex that reducing it to practice for a large and heterologous dataset such as the Influenza sequence database is not practical withtechnology. Overall, the MRCA and COT approaches are impractical forapplication to the complex Influenza sequence database.

A third type of computational analysis is the consensus sequenceapproach. Because the consensus sequence is composed of the amino acidmost commonly observed at each position, it likely represents themost-fit state of the virus. Thus, effective evasion of the immuneresponse by selection of a sequence divergent from consensus may resultin a less fit virus from a replicative standpoint. The consensussequence approach favors heavily sampled sublineages and deemphasizesoutliers. As such, the approaches utilized herein are far morestraightforward than the other types of computational analyses.Furthermore, these approaches can use the entire data set for RSV. Oneadvantage of the consensus sequence is that it minimizes the geneticdifferences between vaccine strains and contemporary isolates,effectively reducing the extent of diversity by half, and thus it mayhave enhanced potential for eliciting cross-reactive responses.

Vaccine Design

To design the vaccine antigen of the present invention, proteinsequences of the F0-, N-, and M2-1-proteins of RSV were retrieved fromthe National Center for Biotechnology Information (NCBI) RSV Resourcedatabase (http://www.ncbi.nlm.nih.gov). Protein sequences were chosenfrom different RSV subtype A strains.

A F0 consensus sequence was derived by alignment of all non-identicalsequences of the F-protein using MUSCLE version 3.6 and applying themajority rule. The vaccine's F0 consensus sequence was designed on thebasis of the alignment of the different RSV sequences. The sequencesimilarity of the vaccine consensus F0 sequence was measured performingBLAST analysis, which stands for Basic Local Alignment Search Tool andis publicly available through the NCBI. The highest average similarityof the consensus sequence, calculated compared to all RSV sequences inthe database, was 100% with respect to the human respiratory syncytialvirus A2 strain.

Further, the vaccine's F0 sequence lacks the transmembrane regionresiding in amino acids 525 to 574 to allow for the secretion of F0ΔTM.

Finally, the vaccine F0ΔTM sequence was codon-optimized for expressionin eukaryotic cells.

The vaccine's N consensus sequence was derived by alignment of allnon-identical sequences of the N-protein using MUSCLE version 3.6 andapplying the majority rule. BLAST analysis of the N consensus sequencefound the best alignment with the human respiratory syncytial virus A2strain. The vaccine's N sequence was then codon-optimized for expressionin eukaryotic cells.

A M2-1 consensus sequence was derived by alignment of all non-identicalsequences of the M2-1-protein using MUSCLE version 3.6 and applying themajority rule. BLAST analysis of the M2-1 consensus sequence found thebest alignment with the human respiratory syncytial virus A2 strain.Finally, the vaccine M2-1 sequence was codon-optimized for expression ineukaryotic cells.

The vaccines F0ΔTM sequence and N sequence were spaced by the cleavagesequence 2A of the Foot and Mouth Disease virus. The vaccines N sequenceand M2-1 sequence were separated by a flexible linker (GGGSGGG; SEQ IDNO: 6).

Finally, the codon-optimized viral genes were cloned as the single openreading frame F0ΔTM-N-M2-1. A schematic diagram of the antigencomposition is given in FIG. 1.

Generation of DNA Plasmids Encoding F0ΔTM and F0ΔTM-N-M2-1

Consensus F0ΔTM, N and M2-1 sequences were optimized for mammalianexpression, including the addition of a Kozak sequence and codonoptimization. The DNA sequence encoding the multi-antigen vaccine waschemically synthesized and then sub-cloned by suitable restrictionenzymes EcoRV and NotI into the pVJTetOCMV shuttle vector under thecontrol of the CMV promoter.

Generation of PanAd3 Viral-Vectored RSV Vaccine

A viral-vectored RSV vaccine PanAd3/F0ΔTM-N-M2-1 was generated whichcontains a 809 aa polyprotein coding for the consensus F0ΔTM, N and M2-1proteins fused by a flexible linker.

Bonobo Adenovirus type 3 (PanAd3) is a novel adenovirus strain withimproved seroprevalence and has been described previously.

Cloning of F0ΔTM-N-M2-1 from the plasmid vector pVJTetOCMV/F0ΔTM-N-M2-1into the PanAd3 pre-Adeno vector was performed by cutting out theantigen sequences flanked by homologous regions and enzymatic in vitrorecombination.

Analysis of Antigen Expression in Mammalian Cells

To control that the unique combination of viral antigens was efficientlyexpressed and correctly processed into mammalian cells, Hela cells weretransfected with 10 μg of DNA plasmid encoding the F0ΔTM-N-M2-1 antigen.Cells were cultured for 36 hours before the supernatant was collectedand cell lysates were prepared. Proteins were separated by SDS-PAGE andblotted onto nylon filters. A mouse monoclonal antibody (mAb8) raisedagainst the M viral protein (gift from Dr. Geraldine Taylor) was used toreveal the expressed proteins.

As shown in FIGS. 2 and 3, the fused viral protein N-M2-1 is veryefficiently released from the polyprotein by the 2A cleavage site andrecognized as a major band by mAb8. Very few high molecular weightprecursor is present at steady-state in the cells. Lysates of Hep2-cellsinfected with RSV strain A were used as control.

Non-Reducing SDS-PAGE and Western blot analysis of the cell culturemedium showed that the F-protein deleted of the trans-membrane region issecreted into the supernatant (see FIG. 3, lane RSV). The molecularweight of the F-protein in the supernatant is consistent withhomotrimeric F-protein, which is its native configuration.

Example 2 Vaccine Immunogenicity in Mice

Anti-F Antibodies by DNA Immunization

DNA plasmids encoding F0ΔTM-N-M2-1 or F0ΔTM alone were used to immunizemice by DNA plasmid injection and electroporation (GET) with a regimenof priming and boosting at three weeks post prime. Sera of immunizedmice were collected two weeks after boosting and pooled.

Supernatants from Hela cells infected with PanAd3/F0ΔTM-N-M2-1 at MOI250 were separated on non-reducing SDS-PAGE, blotted onto nylon filtersand probed with different dilutions of sera from mice immunized withF0ΔTM or F0ΔTM-N-M2-1.

As shown in FIGS. 4A and B, the antibody titers raised by the F-proteinexpressed in the context of the vaccine antigen are at least 30 timeshigher than those elicited by the F-protein alone. Thus, theF0ΔTM-N-M2-1 antigen has superior immunogenic properties in inducingB-cell responses in mice.

T Cell Response

The immunological potency of the chimpanzee adenoviral vector PanAd3bearing the RSV vaccine antigen F0ΔTM-N-M2-1 was evaluated in mice.

Groups of Balb/C mice were immunized by intramuscular injection in thequadriceps with increasing dose of PanAd3/F0ΔTM-N-M2-1. 4 weeks aftervaccination mice were sacrificed and splenocytes were subjected toIFNγ-Elispot assay using mapped immunodominant peptides from RSV F- andM-proteins (peptide GWYTSVITIELSNIKE (F aa 51-66) peptide KYKNAVTEL (Faa 85-93) and peptide SYIGSINNI (M aa 282-290)).

As shown in FIG. 5, a potent T cell response was observed against knownBalb/C immunodominant epitopes against RSV F and M proteins.

Example 3 Induction of Neutralizing Antibodies by H1p

The novel chimeric H1p protein engineered to contain the multibasic HA0cleavage site from H5N1 is efficiently expressed and fully cleaved intransfected HeLa cells. The equivalent protein with the wild typecleavage site, H1, is not cleaved in HeLa cells, as shown in FIG. 7.

In order to control that the chimeric H1p protein is correctly displayedon the cell membrane, a whole-cell FACS binding assay has been performedusing a polyclonal anti-HA serum to reveal the transfected protein onthe cell surface. As shown in FIG. 8, H1p is exposed on the cellmembrane as efficiently as the corresponding wild type HA protein.

To measure the immunological potency of H1p, Balb/C mice were immunizedwith plasmid DNA vectors encoding the modified H1p and the unmodified H1(PVJ-H1p and PVJ-H1, respectively). The sera from immunized animals havebeen analyzed by ELISA on purified recombinant HA protein(H1N1California2009). The anti-HA titers elicited by the engineered H1pprotein were surprisingly higher than those elicited by the HA bearingthe wild type protease cleavage site (FIG. 9).

To confirm and expand these results, the sera from H1 and H1p immunizedanimals were tested for their capacity to neutralize the infection ofretroviral vectors pseudotyped with the Flu HA protein in a cell culturebased assay. Pseudovirions are infectious for a single cycle ofinfection in which they express the reporter gene luciferase. FIG. 10shows the serum neutralization capacity in a HA (H1N1Mexico2009)pseudotyped virus particles infection assay on MDCK cells. The resultconfirms that the antibodies elicited by H1p have greater neutralizingactivity than those induced by H1 protein.

Example 4 Enhanced Antibody Titer by a Polyprotein Comprising NP, M1 andH1p

Head to head comparison of the immunological potency of the H1p andNPM1H1p revealed that the HA protein expressed in the context of thetriple antigen induces higher antibody titer than HA alone. FIG. 11shows the results of an ELISA assay where a recombinant HA(H1N1California 2009) was coated on the bottom of 96 well plate. Serialdilutions of sera of animals immunized with H1p and NPM1H1p were put onthe plate and the bound IgG were revealed with an anti-mouse IgGsecondary antibody. As already observed for the RSV antigen, theco-expression of internal antigens (NP and M1) with the surface exposedantigen (HA) improves the humoral response directed to the latterprotein.

Example 5 Processing of the Novel FLU Antigen Composed of NP, M1 and H1p

To control the expression and processing of the vaccine antigenproteins, HeLa cells have been transfected with an expression plasmidcontaining NPM1H1p under the control of the CMV promoter. Cells wereharvested 48 hours after transfection. Half cells were lysed for WesternBlot analysis (FIG. 12) and half were incubated with a commerciallyavailable antibody C179 (Okuno Y, JVI 1994), which binds the stem regionof the HA protein (FIG. 13) and analysed by FACS. Western blot analysisof the total cell lysate shows a unique 70 kDa band which correspond tothe NPM1 fusion protein (FIG. 12). This indicates that the antigen isfully and correctly processed out of the 2A cleavage site. FIG. 13 showsthat the released H1p protein is then displayed on the cell membrane andcorrectly folded, as detected by the use of a conformation-dependentantibody C179 which binds to the HA stem region. Accordingly, the novelFLU antigen composed of NP, M1 and H1p is correctly processed and thereleased HA protein is displayed on the cell surface and recognized by aconformational antibody.

REFERENCES

-   1) Ono & Freed, (2005), Adv. Virus Res., 273:5419-5442-   2) Collins P et al., (1996). Parainfluenza viruses, 1205-1241. In    Fields et al. (ed.), Fields Virology. Lippincot-Raven Publishers,    Philadelphia (Pa.) USA-   3) Shay D K et al., (1999), Bronchiolitis-Associated    Hospitalizations Among US Children, 1980-1996. JAMA 282:1440-1446-   4) Simoes E A & Carbonell-Estrany X (2003), Pediatr Infect Dis J    22:S13-8; discussion S18-20.-   5) Collins P L & Graham B S (2008), J Virol 82:2040-55-   6) Falsey A R et al., (2005), N Engl J Med 352:1749-59-   7) Fleming D M & Elliot A J (2007), Eur Respir J 30:1029-31-   8) Cardenas S et al., (2005), Expert Rev Anti Infect Ther 3:719-26-   9) Kim H W et al., (1969), Amer J Epidemiol 89:422-434-   10) Delgado M F et al., (2009), Nat Med 15:34-41-   11) Graham B S et al., (1993), J Immunol 151:2032-2040-   12) Polack F P et al., (2002), J Exp Med 196:859-865-   13) Castilow E M & Varga S M, (2008), Future Virol 3:445-454-   14) Hansen S et al., (2011), Nature 473:523-527-   15) Flatz, L et al. (2010), Nature Med 16:339-345

Sequence Listing Free Text Information

-   -   SEQ ID NO: 1 F protein minimal sequence    -   SEQ ID NO: 2 F0ΔTM    -   SEQ ID NO: 3 N protein minimal sequence    -   SEQ ID NO: 4 N protein of RSV    -   SEQ ID NO: 5 M2-1 protein of RSV    -   SEQ ID NO: 6 peptide linker    -   SEQ ID NO: 7 F0ΔTM-N-M2-1    -   SEQ ID NO: 8 HA subtype H1 consensus sequence    -   SEQ ID NO: 9 HA subtype H1 consensus sequence modified H0        cleavage site    -   SEQ ID NO: 10 HA subtype H5 consensus sequence    -   SEQ ID NO: 11 NP consensus sequence    -   SEQ ID NO: 12 M1 consensus sequence    -   SEQ ID NO: 13 NP-M1-HA (amino acid sequence)    -   SEQ ID NO: 14 NP-M1-HA (nucleic acid sequence)    -   SEQ ID NO: 15 H0 cleavage site sequence    -   SEQ ID NO: 16 H0 cleavage site sequence    -   SEQ ID NO: 17 H0 cleavage site sequence    -   SEQ ID NO: 18 H0 cleavage site sequence    -   SEQ ID NO: 19 H0 cleavage site sequence    -   SEQ ID NO: 20 HA subtype H3 strain A/Wellington/01/2004(H3N2)    -   SEQ ID NO: 21 HA subtype H3 strain A/Wellington/01/2004(H3N2)        modified H0 cleavage site

1. An expression system comprising polynucleotides encoding proteins,wherein the expression system comprises a first polynucleotide encodingat least one protein, peptide or variant thereof, which induces a T cellresponse, and a second polynucleotide encoding at least one proteinpeptide or variant thereof, which induces an anti-pathogenic B cellresponse.
 2. The expression system of claim 1 wherein the firstpolynucleotide and the second polynucleotide are linked such that theyare expressed as an artificial polyprotein.
 3. The expression system ofclaim 1, wherein the proteins, peptides or fragments encoded by thefirst and the second polynucleotide are separated co- orposttranslationally.
 4. The expression system of claim 1, wherein apolynucleotide which encodes a cleavage site is positioned between thefirst polynucleotide and the second polynucleotide.
 5. The expressionsystem of claim 4, wherein the cleavage site is a self-cleaving site oran endopeptidase cleavage site.
 6. (canceled)
 7. The expression-systemof claim 1, further comprising a third polynucleotide encoding a proteinor variant thereof. 8.-13. (canceled)
 14. The expression system of claim1, wherein the expression system comprises at least one vector selectedfrom the group consisting of plasmid vectors, cosmid vectors, phagevectors, and viral vectors, viral like particles, and bacterial spores.15. The expression-system of claim 14, wherein the viral vector is anadenovirus vector selected from the group consisting of PanAd1, PanAd2,PanAd3, ChAd55, ChAd 73, ChAd83, ChAd146, ChAd147, ChAd3, ChAd4, ChAd5,ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19,ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44,ChAd63 and ChAd82.
 16. The expression-system of claim 14, wherein theviral vector is selected from the group consisting of cytomegalovirusevectors, arena virus vectors, and pox virus vectors.
 17. The expressionsystem of claim 1, wherein at least one of the first polynucleotide orthe second polynucleotide is selected from the group consisting of aprotein, peptide or variant thereof from a pathogen.
 18. The expressionsystem of claim 17, wherein the pathogen is a virus.
 19. The expressionsystem of claim 1, wherein the protein, that induces a T cell responseis a non-structural and/or internal protein of a virus, and/or theprotein that induces an anti-pathogenic B cell response is a structuraland/or surface protein of a virus. 20.-23. (canceled)
 24. The expressionsystem of claim 18, wherein the virus is selected from the groupconsisting of paramyxoviruses and orthomyxoviruses. 25.-26. (canceled)27. The expression system of claim 24, wherein the paramyxovirus isselected from the group consisting of Pneumovirus, Metapneumovirus, andRespirovirus.
 28. (canceled)
 29. The expression system of claim 19,wherein the structural and/or surface protein of the virus is selectedfrom the group of paramyxovirus proteins consisting of fusion protein(F), and any of the attachment glycoproteins G, H, and HN. 30.-31.(canceled)
 32. The expression system of claim 1, wherein thenon-structural and/or internal protein is selected from the group ofparamyxovirus proteins consisting of nucleoprotein N, Matrix proteins Mand M2, Phosphoprotein P, non structural proteins NS1 and NS2, and thecatalytic subunit of the polymerase (L). 33.-35. (canceled)
 36. Theexpression system of claim 24, wherein the orthomyxovirus is selectedfrom Influenza A virus, Influenza B virus, Influenza C virus,Thogotovirus, Isavirus and unclassified Orthomyxoviridae.
 37. (canceled)38. The expression system of claim 19, wherein the structural and/orsurface protein of the virus is selected from the group orthomyxovirusproteins consisting of hemagglutinin (HA) and neuraminidase (NA).39.-40. (canceled)
 41. The expression system of claim 19, wherein thenon-structural and/or internal protein(s) is selected from the group oforthomyxovirus proteins consisting of nucleoprotein (NP), matrixprotein1 (MP1), matrixprotein M2, non-structural protein 1 (NS1),non-structural protein2/nuclear export protein (NS2/NEP), polymerasesubunit protein PA, polymerase subunit protein PB1, polymerase subunitprotein PB2, and PB1-F2 protein encoded by an alternate reading frame inthe PB1 gene. 42.-43. (canceled)
 44. An isolated protein mixture encodedby the expression system of claim
 1. 45. An isolated host cellcontaining the expression-system according to claim
 1. 46. A compositioncomprising the expression-system according to claim 1 and apharmaceutically acceptable carrier and/or excipient. 47.-50. (canceled)51. A method of treatment or prevention of a viral disease comprisingadministration of an effective amount of the expression-system accordingto claim
 1. 52. The method of claim 51 wherein the disease is caused bya virus selected from the group consisting of cytomegalovirus, arenavirus, pox virus, paramyxovirus, and orthomyxovirus.
 53. (canceled) 54.A nucleic acid construct which comprises a polynucleotide encoding amodified influenza hemagglutinin (HA), wherein the HA0 cleavage site ismodified by introducing one or more basic amino acids. 55.-57.(canceled)
 58. The expression system of claim 1, comprising at least onepolynucleotide encoding a modified influenza HA protein, wherein the HA0cleavage site is modified by introducing one or more basic amino acids.59. An isolated protein mixture encoded by the expression system ofclaim
 58. 60. An isolated host cell containing the expression-systemaccording to claim
 58. 61. A composition comprising theexpression-system of claim 58 and a pharmaceutically acceptable carrierand/or excipient. 62.-63. (canceled)
 64. A method of treatment orprevention of a viral disease comprising administration of an effectiveamount of the expression-system according to claim
 586. 65. (canceled)